Journal articles: 'SfGFP, superfolder green fluorescent protein'

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Pedelacq, Jean-Denis, and Stéphanie Cabantous. "Development and Applications of Superfolder and Split Fluorescent Protein Detection Systems in Biology." International Journal of Molecular Sciences 20, no. 14 (July 15, 2019): 3479. http://dx.doi.org/10.3390/ijms20143479.

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Molecular engineering of the green fluorescent protein (GFP) into a robust and stable variant named Superfolder GFP (sfGFP) has revolutionized the field of biosensor development and the use of fluorescent markers in diverse area of biology. sfGFP-based self-associating bipartite split-FP systems have been widely exploited to monitor soluble expression in vitro, localization, and trafficking of proteins in cellulo. A more recent class of split-FP variants, named « tripartite » split-FP, that rely on the self-assembly of three GFP fragments, is particularly well suited for the detection of protein–protein interactions. In this review, we describe the different steps and evolutions that have led to the diversification of superfolder and split-FP reporter systems, and we report an update of their applications in various areas of biology, from structural biology to cell biology.

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Olenginski, Gregory M., Juliana Piacentini, Darcy R. Harris, Nicolette A. Runko, Brianna M. Papoutsis, Jordan R. Alter, Kenneth R. Hess, Scott H. Brewer, and Christine M. Phillips-Piro. "Structural and spectrophotometric investigation of two unnatural amino-acid altered chromophores in the superfolder green fluorescent protein." Acta Crystallographica Section D Structural Biology 77, no. 8 (July 29, 2021): 1010–18. http://dx.doi.org/10.1107/s2059798321006525.

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The spectrophotometric properties of the green fluorescent protein (GFP) result from the post-translationally cyclized chromophore composed of three amino acids including a tyrosine at the center of the β-barrel protein. Altering the amino acids in the chromophore or the nearby region has resulted in numerous GFP variants with differing photophysical properties. To further examine the effect of small atomic changes in the chromophore on the structure and photophysical properties of GFP, the hydroxyl group of the chromophore tyrosine was replaced with a nitro or a cyano group. The structures and spectrophotometric properties of these superfolder GFP (sfGFP) variants with the unnatural amino acids (UAAs) 4-nitro-L-phenylalanine or 4-cyano-L-phenylalanine were explored. Notably, the characteristic 487 nm absorbance band of wild-type (wt) sfGFP is absent in both unnatural amino-acid-containing protein constructs (Tyr66pNO2Phe-sfGFP and Tyr66pCNPhe-sfGFP). Consequently, neither Tyr66pNO2Phe-sfGFP nor Tyr66pCNPhe-sfGFP exhibited the characteristic emission of wt sfGFP centered at 511 nm when excited at 487 nm. Tyr66pNO2Phe-sfGFP appeared orange due to an absorbance band centered at 406 nm that was not present in wt sfGFP, while Tyr66pCNPhe-sfGFP appeared colorless with an absorbance band centered at 365 nm. Mass spectrometry and X-ray crystallography confirmed the presence of a fully formed chromophore and no significant structural changes in either of these UAA-containing protein constructs, signaling that the change in the observed photophysical properties of the proteins is the result of the presence of the UAA in the chromophore.

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Maurici, Nicole, Nicole Savidge, Byung Uk Lee, Scott H. Brewer, and Christine M. Phillips-Piro. "Crystal structures of green fluorescent protein with the unnatural amino acid 4-nitro-L-phenylalanine." Acta Crystallographica Section F Structural Biology Communications 74, no. 10 (September 19, 2018): 650–55. http://dx.doi.org/10.1107/s2053230x1801169x.

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The X-ray crystal structures of two superfolder green fluorescent protein (sfGFP) constructs containing a genetically incorporated spectroscopic reporter unnatural amino acid, 4-nitro-L-phenylalanine (pNO2F), at two unique sites in the protein have been determined. Amber codon-suppression methodology was used to site-specifically incorporate pNO2F at a solvent-accessible (Asp133) and a partially buried (Asn149) site in sfGFP. The Asp133pNO2F sfGFP construct crystallized with two molecules per asymmetric unit in space group P3221 and the crystal structure was refined to 2.05 Å resolution. Crystals of Asn149pNO2F sfGFP contained one molecule of sfGFP per asymmetric unit in space group P4122 and the structure was refined to 1.60 Å resolution. The alignment of Asp133pNO2F or Asn149pNO2F sfGFP with wild-type sfGFP resulted in small root-mean-square deviations, illustrating that these residues do not significantly alter the protein structure and supporting the use of pNO2F as an effective spectroscopic reporter of local protein structure and dynamics.

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Lajoie, Patrick, Robyn D. Moir, Ian M. Willis, and Erik L. Snapp. "Kar2p availability defines distinct forms of endoplasmic reticulum stress in living cells." Molecular Biology of the Cell 23, no. 5 (March 2012): 955–64. http://dx.doi.org/10.1091/mbc.e11-12-0995.

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Accumulation of misfolded secretory proteins in the endoplasmic reticulum (ER) activates the unfolded protein response (UPR) stress pathway. To enhance secretory protein folding and promote adaptation to stress, the UPR upregulates ER chaperone levels, including BiP. Here we describe chromosomal tagging of KAR2, the yeast homologue of BiP, with superfolder green fluorescent protein (sfGFP) to create a multifunctional endogenous reporter of the ER folding environment. Changes in Kar2p-sfGFP fluorescence levels directly correlate with UPR activity and represent a robust reporter for high-throughput analysis. A novel second feature of this reporter is that photobleaching microscopy (fluorescence recovery after photobleaching) of Kar2p-sfGFP mobility reports on the levels of unfolded secretory proteins in individual cells, independent of UPR status. Kar2p-sfGFP mobility decreases upon treatment with tunicamycin or dithiothreitol, consistent with increased levels of unfolded proteins and the incorporation of Kar2p-sfGFP into slower-diffusing complexes. During adaptation, we observe a significant lag between down-regulation of the UPR and resolution of the unfolded protein burden. Finally, we find that Kar2p-sfGFP mobility significantly increases upon inositol withdrawal, which also activates the UPR, apparently independent of unfolded protein levels. Thus Kar2p mobility represents a powerful new tool capable of distinguishing between the different mechanisms leading to UPR activation in living cells.

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Jahangir, ZMG Sarwar, and Arleta Helena Marnik. "A study protocol to prepare an RBD protein for vaccine against COVID-19." F1000Research 10 (September 20, 2021): 943. http://dx.doi.org/10.12688/f1000research.54738.1.

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Background: The SARS-CoV-2 pandemic is a global threat to humans and the world’s economy. Effective and safe vaccines against this virus are essential to control and eradicate the pandemic. The currently applied vaccines carry SARS-CoV-2 spike-protein mRNA/cDNA. These vaccines go through several cellular processes in the recipients for producing antigens. On the contrary, the SARS-CoV-2 RBD (receptor binding domain)-protein is an antigen. It will directly stimulate antibody production against SARS-CoV-2. Hence, we propose to produce SARS-CoV-2 RBD-protein as a fast acting, effective and safe vaccine. Methods: We propose to reconstruct a plasmid carrying three types of DNA sequences: RBD cDNA, FP (fusion peptide) DNA and sfGFP(superfolder green fluorescent protein), cDNA creating the RBD-FP-sfGFP DNA within an orf (open reading frame). Escherichia coli, C2566H, transformed with the reconstructed plasmid will express RBD-FP-sfGFP fusion protein producing green fluorescent cfu (colony forming unit). The RBD-protein will be separated from the sfGFP using an FP specific enterokinase, and eluted by HIC (hydrophobic interaction chromatography), detected with a BioVision Elisa kit, and quantified by spectrophotometry at UV280nm. Results: The plasmid reconstruct will carry ampr (ampicillin-resistant) gene as a selective marker and a T7 promoter controlling the expression of RBD-FP-sfGFP fusion protein. The transformed Escherichia coli will efficiently express the RBD-FP-sfGFP fusion protein. The highly efficient sfGFP fused within the RBD-FP-sfGFP will produce green fluorescent cfu. The RBD-FP-sfGFP protein extract from the green cfu, digested by enterokinase and separated by the HIC will produce pure RBD protein. Conclusion: A positive BioVision ELISA test detects <10 pg RBD protein/ml of the sample. A larger sample of the purified RBD protein can be used as a vaccine following a standard formulation and safety protocols. Once administered, the RBD protein will stimulate antibody production against the SARS-CoV-2 virus. The RBD protein has no potential to recombine with human genome.

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Ramadani-Muja, Jeta, Benjamin Gottschalk, Katharina Pfeil, Sandra Burgstaller, Thomas Rauter, Helmut Bischof, Markus Waldeck-Weiermair, Heiko Bugger, Wolfgang F. Graier, and Roland Malli. "Visualization of Sirtuin 4 Distribution between Mitochondria and the Nucleus, Based on Bimolecular Fluorescence Self-Complementation." Cells 8, no. 12 (December 6, 2019): 1583. http://dx.doi.org/10.3390/cells8121583.

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Mitochondrial sirtuins (Sirts) control important cellular processes related to stress. Despite their regulatory importance, however, the dynamics and subcellular distributions of Sirts remain debatable. Here, we investigate the subcellular localization of sirtuin 4 (Sirt4), a sirtuin variant with a mitochondrial targeting sequence (MTS), by expressing Sirt4 fused to the superfolder green fluorescent protein (Sirt4-sfGFP) in HeLa and pancreatic β-cells. Super resolution fluorescence microscopy revealed the trapping of Sirt4-sfGFP to the outer mitochondrial membrane (OMM), possibly due to slow mitochondrial import kinetics. In many cells, Sirt4-sfGFP was also present within the cytosol and nucleus. Moreover, the expression of Sirt4-sfGFP induced mitochondrial swelling in HeLa cells. In order to bypass these effects, we applied the self-complementing split fluorescent protein (FP) technology and developed mito-STAR (mitochondrial sirtuin 4 tripartite abundance reporter), a tripartite probe for the visualization of Sirt4 distribution between mitochondria and the nucleus in single cells. The application of mito-STAR proved the importation of Sirt4 into the mitochondrial matrix and demonstrated its localization in the nucleus under mitochondrial stress conditions. Moreover, our findings highlight that the self-complementation of split FP is a powerful technique to study protein import efficiency in distinct cellular organelles.

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Gao, Wei, Ning Bu, and Yuan Lu. "Efficient Incorporation of Unnatural Amino Acids into Proteins with a Robust Cell-Free System." Methods and Protocols 2, no. 1 (February 12, 2019): 16. http://dx.doi.org/10.3390/mps2010016.

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Unnatural proteins are crucial biomacromolecules and have been widely applied in fundamental science, novel biopolymer materials, enzymes, and therapeutics. Cell-free protein synthesis (CFPS) system can serve as a robust platform to synthesize unnatural proteins by highly effective site-specific incorporation of unnatural amino acids (UNAAs), without the limitations of cell membrane permeability and the toxicity of unnatural components. Here, we describe a quick and simple method to synthesize unnatural proteins in CFPS system based on Escherichia coli crude extract, with unnatural orthogonal aminoacyl-tRNA synthetase and suppressor tRNA evolved from Methanocaldococcus jannaschii. The superfolder green fluorescent protein (sfGFP) and p-propargyloxyphenylalanine (pPaF) were used as the model protein and UNAA. The synthesis of unnatural sfGFPs was characterized by microplate spectrophotometer, affinity chromatography, and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). This protocol provides a detailed procedure guiding how to use the powerful CFPS system to synthesize unnatural proteins on demand.

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Subach, Oksana M., and Fedor V. Subach. "GAF-CaMP3–sfGFP, An Enhanced Version of the Near-Infrared Genetically Encoded Positive Phytochrome-Based Calcium Indicator for the Visualization of Neuronal Activity." International Journal of Molecular Sciences 21, no. 18 (September 19, 2020): 6883. http://dx.doi.org/10.3390/ijms21186883.

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The first generation of near-infrared, genetically encoded calcium indicators (NIR-GECIs) was developed from bacterial phytochrome-based fluorescent proteins that utilize biliverdin (BV) as the chromophore moiety. However, NIR-GECIs have some main drawbacks such as either an inverted response to calcium ions (in the case of NIR-GECO1) or a limited dynamic range and a lack of data about their application in neurons (in the case of GAF-CaMP2–superfolder green fluorescent protein (sfGFP)). Here, we developed an enhanced version of the GAF-CaMP2–sfGFP indicator, named GAF-CaMP3–sfGFP. The GAF-CaMP3–sfGFP demonstrated spectral characteristics, molecular brightness, and a calcium affinity similar to the respective characteristics for its progenitor, but a 2.9-fold larger DF/F response to calcium ions. As compared to GAF-CaMP2–sfGFP, in cultured HeLa cells, GAF-CaMP3–sfGFP had similar brightness but a 1.9-fold larger DF/F response to the elevation of calcium ions levels. Finally, we successfully utilized the GAF-CaMP3–sfGFP for the monitoring of the spontaneous and stimulated activity of neuronal cultures and compared its performance with the R-GECO1 indicator using two-color confocal imaging. In the cultured neurons, GAF-CaMP3–sfGFP showed a linear DF/F response in the range of 0–20 APs and in this range demonstrated a 1.4-fold larger DF/F response but a 1.3- and 2.4-fold slower rise and decay kinetics, respectively, as compared to the same parameters for the R-GECO1 indicator.

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Dippel, Andrew B., Gregory M. Olenginski, Nicole Maurici, Melanie T. Liskov, Scott H. Brewer, and Christine M. Phillips-Piro. "Probing the effectiveness of spectroscopic reporter unnatural amino acids: a structural study." Acta Crystallographica Section D Structural Biology 72, no. 1 (January 1, 2016): 121–30. http://dx.doi.org/10.1107/s2059798315022858.

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The X-ray crystal structures of superfolder green fluorescent protein (sfGFP) containing the spectroscopic reporter unnatural amino acids (UAAs) 4-cyano-L-phenylalanine (pCNF) or 4-ethynyl-L-phenylalanine (pCCF) at two unique sites in the protein have been determined. These UAAs were genetically incorporated into sfGFP in a solvent-exposed loop region and/or a partially buried site on the β-barrel of the protein. The crystal structures containing the UAAs at these two sites permit the structural implications of UAA incorporation for the native protein structure to be assessed with high resolution and permit a direct correlation between the structure and spectroscopic data to be made. The structural implications were quantified by comparing the root-mean-square deviation (r.m.s.d.) between the crystal structure of wild-type sfGFP and the protein constructs containing either pCNF or pCCF in the local environment around the UAAs and in the overall protein structure. The results suggest that the selective placement of these spectroscopic reporter UAAs permits local protein environments to be studied in a relatively nonperturbative fashion with site-specificity.

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Khmelinskii, Anton, Matthias Meurer, Chi-Ting Ho, Birgit Besenbeck, Julia Füller, Marius K. Lemberg, Bernd Bukau, Axel Mogk, and Michael Knop. "Incomplete proteasomal degradation of green fluorescent proteins in the context of tandem fluorescent protein timers." Molecular Biology of the Cell 27, no. 2 (January 15, 2016): 360–70. http://dx.doi.org/10.1091/mbc.e15-07-0525.

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Tandem fluorescent protein timers (tFTs) report on protein age through time-dependent change in color, which can be exploited to study protein turnover and trafficking. Each tFT, composed of two fluorescent proteins (FPs) that differ in maturation kinetics, is suited to follow protein dynamics within a specific time range determined by the maturation rates of both FPs. So far, tFTs have been constructed by combining slower-maturing red fluorescent proteins (redFPs) with the faster-maturing superfolder green fluorescent protein (sfGFP). Toward a comprehensive characterization of tFTs, we compare here tFTs composed of different faster-maturing green fluorescent proteins (greenFPs) while keeping the slower-maturing redFP constant (mCherry). Our results indicate that the greenFP maturation kinetics influences the time range of a tFT. Moreover, we observe that commonly used greenFPs can partially withstand proteasomal degradation due to the stability of the FP fold, which results in accumulation of tFT fragments in the cell. Depending on the order of FPs in the timer, incomplete proteasomal degradation either shifts the time range of the tFT toward slower time scales or precludes its use for measurements of protein turnover. We identify greenFPs that are efficiently degraded by the proteasome and provide simple guidelines for the design of new tFTs.

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Shen, Jing, Wenlu Zhang, Chunyang Gan, Xiafei Wei, Jie Li, Yuxue Sun, Yi Yuan, et al. "Strategies to improve the fluorescent signal of the tripartite sfGFP system." Acta Biochimica et Biophysica Sinica 52, no. 9 (June 24, 2020): 998–1006. http://dx.doi.org/10.1093/abbs/gmaa073.

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Abstract Bimolecular fluorescence complementation (BiFC) is a popular method used to detect protein–protein interactions. For a BiFC assay, a fluorescent protein is usually split into two parts, and the fluorescence is recovered upon the interaction between the fused proteins of interest. As an elegant extension of BiFC, a tripartite superfold green fluorescent protein (sfGFP) system that has the advantages of low background fluorescence and small fusion tag size has been developed. However, the tripartite system exhibits a low fluorescence signal in some cases. To address this problem, we proposed to increase the affinity between the two parts, G1–9 and G11, of the tripartite system by adding affinity pairs. Among the three affinity pairs tested, LgBiT-HiBiT improved both the signal and signal-to-noise (S/N) ratio to the greatest extent. More strikingly, the direct covalent fusion of G11 to G1–9, which converted the tripartite system into a new bipartite system, enhanced the S/N ratio from 20 to 146, which is superior to the bipartite sfGFP system split at 157/158 or 173/174. Our results implied that the 10th β-strand of sfGFP has a low affinity and a good recovery efficiency to construct a robust BiFC system, and this concept might be applied to other fluorescent proteins with similar structure to construct new BiFC systems.

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Wu, Xudong, Di Wu, Zhisheng Lu, Wentao Chen, Xiaojian Hu, and Yu Ding. "A Novel Method for High-Level Production of TEV Protease by Superfolder GFP Tag." Journal of Biomedicine and Biotechnology 2009 (2009): 1–8. http://dx.doi.org/10.1155/2009/591923.

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Because of its stringent sequence specificity, tobacco etch virus (TEV) protease is widely used to remove fusion tags from recombinant proteins. Due to the poor solubility of TEV protease, many strategies have been employed to increase the expression level of this enzyme. In our work, we introduced a novel method to produce TEV protease by using visible superfolder green fluorescent protein (sfGFP) as the fusion tag. The soluble production and catalytic activity of six variants ofsfGFP-TEV was examined, and then the best variant was selected for large-scale production. After purified by Ni-NTA affinity chromatography and Q anion exchange chromatography, the best variant ofsfGFP-TEV fusion protease was obtained with purity of over 98% and yield of over 320 mg per liter culture. ThesfGFP-TEV had a similar catalytic activity to that of the original TEV protease. Our research showed a novel method of large-scale production of visible and functional TEV protease for structural genomics research and other applications.

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Bai, Chaoxian, Yang Zhang, Xuejin Zhao, Yiling Hu, Sihai Xiang, Jin Miao, Chunbo Lou, and Lixin Zhang. "Exploiting a precise design of universal synthetic modular regulatory elements to unlock the microbial natural products in Streptomyces." Proceedings of the National Academy of Sciences 112, no. 39 (September 15, 2015): 12181–86. http://dx.doi.org/10.1073/pnas.1511027112.

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There is a great demand for precisely quantitating the expression of genes of interest in synthetic and systems biotechnology as new and fascinating insights into the genetics of streptomycetes have come to light. Here, we developed, for the first time to our knowledge, a quantitative method based on flow cytometry and a superfolder green fluorescent protein (sfGFP) at single-cell resolution in Streptomyces. Single cells of filamentous bacteria were obtained by releasing the protoplasts from the mycelium, and the dead cells could be distinguished from the viable ones by propidium iodide (PI) staining. With this sophisticated quantitative method, some 200 native or synthetic promoters and 200 ribosomal binding sites (RBSs) were characterized in a high-throughput format. Furthermore, an insulator (RiboJ) was recruited to eliminate the interference between promoters and RBSs and improve the modularity of regulatory elements. Seven synthetic promoters with gradient strength were successfully applied in a proof-of-principle approach to activate and overproduce the cryptic lycopene in a predictable manner in Streptomyces avermitilis. Our work therefore presents a quantitative strategy and universal synthetic modular regulatory elements, which will facilitate the functional optimization of gene clusters and the drug discovery process in Streptomyces.

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Liauw, Pasqual, Daniela Kannchen, Raphael Gasper, Nina Dyczmons-Nowaczyk, Marc M. Nowaczyk, and Eckhard Hofmann. "Cloning, expression, crystallization and preliminary X-ray studies of a superfolder GFP fusion of cyanobacterial Psb32." Acta Crystallographica Section F Structural Biology Communications 71, no. 4 (March 20, 2015): 409–13. http://dx.doi.org/10.1107/s2053230x15003970.

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A fusion of Psb32 from the thermophilic cyanobacteriumThermosynechococcus elongatusBP-1 (TePsb32) with superfolder GFP was created for enhanced solubility and improved detection and purification. The fusion protein readily formed large hexagonal crystals belonging to space groupP6122. A full data set extending to 2.3 Å resolution was collected at the Swiss Light Source. The phase problem could be solved by using only the sfGFP fusion partner or by using GFP andAtTLP18.3 fromArabidopsis thalianaas search models. Based on this expression construct, a versatile library of 24 vectors combining four different superfolder GFP variants and three affinity tags was generated to facilitate expression and screening of fluorescent fusion proteins.

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Valbuena, Fernando M., Ivy Fitzgerald, Rita L. Strack, Neal Andruska, Luke Smith, and Benjamin S. Glick. "A photostable monomeric superfolder green fluorescent protein." Traffic 21, no. 8 (June 16, 2020): 534–44. http://dx.doi.org/10.1111/tra.12737.

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Dinh, T., and T. G. Bernhardt. "Using Superfolder Green Fluorescent Protein for Periplasmic Protein Localization Studies." Journal of Bacteriology 193, no. 18 (July 15, 2011): 4984–87. http://dx.doi.org/10.1128/jb.00315-11.

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Pédelacq, Jean-Denis, Stéphanie Cabantous, Timothy Tran, Thomas C. Terwilliger, and Geoffrey S. Waldo. "Engineering and characterization of a superfolder green fluorescent protein." Nature Biotechnology 24, no. 1 (December 20, 2005): 79–88. http://dx.doi.org/10.1038/nbt1172.

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Pe´delacq, Jean-Denis, Stéphanie Cabantous, Timothy Tran, Thomas C. Terwilliger, and Geoffrey S. Waldo. "Correction: Corrigendum: Engineering and characterization of a superfolder green fluorescent protein." Nature Biotechnology 24, no. 9 (September 1, 2006): 1170. http://dx.doi.org/10.1038/nbt0906-1170d.

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Nguyen, Nam Hoai, Trang Thi Phuong Phan, Thuoc Linh Tran, and Hoang Duc Nguyen. "Cloning and investigating GFP (green fluorescent protein) allowing higher expression in Bacillus subtilis." Science and Technology Development Journal 18, no. 2 (June 30, 2015): 46–55. http://dx.doi.org/10.32508/stdj.v18i2.1142.

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Gfp gene coding for protein GFP (green fluorescent protein) from the jellyfish Aequorea victoria is the most popular indicator protein, due to (i) easy to recognize the expression through the fluorescent ability, (ii) without substrate or cofactor, (iii) less impact on the fusion protein target form. A lot of modifications of GFP have been studied and improved, in which GFP superfolder containing beneficial properties such as rapid folding, structural stability and strong fluorescence showed great applicability. However, GFP superfolder has just been developed in Escherichia coli. It is difficult to apply this version for all other strains since codon usage and the folding ability of each host species. In this study, with the aim of generating a GFP gene which is suitable for Bacillus subtilis, we mutated in the gene gfp+ to delete general BamHI, XhoI restriction enzyme site for facilitating in cloning step; as well asmodified Y39N, N105T, I171V codon that is compatible with B. subtilis. The results showed that the mutations have made the new version of GFP stronger in fluorescence and sequences of restriction enzymes have also been removed.

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Zhou, Jun, Jian Lin, Cuihong Zhou, Xiaoyan Deng, and Bin Xia. "An improved bimolecular fluorescence complementation tool based on superfolder green fluorescent protein." Acta Biochimica et Biophysica Sinica 43, no. 3 (January 27, 2011): 239–44. http://dx.doi.org/10.1093/abbs/gmq128.

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Slocum, Joshua D., and Lauren J. Webb. "A Double Decarboxylation in Superfolder Green Fluorescent Protein Leads to High Contrast Photoactivation." Journal of Physical Chemistry Letters 8, no. 13 (June 12, 2017): 2862–68. http://dx.doi.org/10.1021/acs.jpclett.7b01101.

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Al-Homsi, Lamis, Souad Al-Okla, and Abdul Q. Abbady. "Preparation of Specific Polyclonal Antibody Against the Recombinant Mutacin Produced by sfGFP Fusion Protein Technology." Open Microbiology Journal 9, no. 1 (July 31, 2015): 70–80. http://dx.doi.org/10.2174/1874285801509010070.

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Mutacin I, a bacteriocin produced bystreptococcus mutans, displays an antimicrobial activity against many gram positive and some gram negative bacteria. Because of its medical importance, production of this short peptide in large scale for future applications is a significant challenge. This work described the improvement of a novel system to produce the recombinant mutacin using fusion protein technology.The short peptide was expressed directly as a fusion protein with a superfolder form of the green florescent protein (sfGFP), resulting in a high yield expression of solublesfGFP-mutacin fusion protein (30 kDa) in the cytoplasm of E. coli. Mutacin was released from the fusion by enzymatic cleavage at the tobacco etch virus (TEV) protease recognition site and separated from the carriersfGFP by nickel affinity and gel filtration chromatography. An additional advantage of this fusion system was tested in the generation of mutacin-specific polyclonal antibodies. Specific anti-mutacin IgGs were affinity purified, and were able to recognize the mutacin-sfGFP fusion protein or the cleaved forms of mutacin.Even though it was efficiently produced (25 mg/L) by this method, pure mutacin was devoid of antibiotic activity. Fourier transform infrared spectroscopy (FTIR) analysis revealed the absence of thioether bonds in the purified mutacin, which are critical for final structure and function of this antibiotic. Determining whether the activity of pure mutacin could be recovered by the reformation of such structures by chemical reaction needs more investigations. The development of this system will provide large quantities of mutacin for future studies and applications as broad spectrum antibacterial peptide.

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Tansila, Natta, Kristian Becker, Chartchalerm Isarankura Na-Ayudhya, Virapong Prachayasittikul, and Leif Bülow. "Metal ion accessibility of histidine-modified superfolder green fluorescent protein expressed in Escherichia coli." Biotechnology Letters 30, no. 8 (March 13, 2008): 1391–96. http://dx.doi.org/10.1007/s10529-008-9692-7.

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Bose, Jeffrey L., Paul D. Fey, and Kenneth W. Bayles. "Genetic Tools To Enhance the Study of Gene Function and Regulation in Staphylococcus aureus." Applied and Environmental Microbiology 79, no. 7 (January 25, 2013): 2218–24. http://dx.doi.org/10.1128/aem.00136-13.

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ABSTRACTThebursa aurealistransposon has been used to create transposon insertion libraries ofBacillus anthracisandStaphylococcus aureus. To provide a set of genetic tools to enhance the utility of these libraries, we generated an allelic-exchange system that allows for the replacement of the transposon with useful genetic markers and fluorescent reporter genes. These tools were tested in the Nebraska Transposon Mutant Library (NTML), containing defined transposon insertions in 1,952 nonessentialS. aureusgenes. First, we generated a plasmid that allows researchers to replace the genes encoding green fluorescent protein (GFP) and erythromycin resistance in the transposon with a noncoding DNA fragment, leaving a markerless mutation within the chromosome. Second, we produced allelic-exchange plasmids to replace the transposon with alternate antibiotic resistance cassettes encoding tetracycline, kanamycin, and spectinomycin resistance, allowing for the simultaneous selection of multiple chromosomal mutations. Third, we generated a series of fluorescent reporter constructs that, after allelic exchange, generate transcriptional reporters encoding codon-optimized enhanced cyan fluorescent protein (ECFP), enhanced yellow fluorescent protein (EYFP), DsRed.T3(DNT), and eqFP650, as well as superfolder green fluorescent protein (sGFP). Overall, combining the NTML with this allelic-exchange system provides an unparalleled resource for the study ofS. aureus.

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Sakhabeev, R. G., D. S. Polyakov, A. D. Goshina, A. A. Vishnya, I. V. Kudryavtsev, E. S. Sinitcina, V. А. Korzhikov-Vlakh, T. B. Tennikova, and M. M. Shavlovsky. "Enhancing the specific T cell immune response against micro- and nanoparticle immobilized antigen." Russian Journal of Infection and Immunity 11, no. 4 (September 20, 2021): 777–83. http://dx.doi.org/10.15789/2220-7619-ets-1374.

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The current study was a part of the project on generating viral particle traps occurring due to covalent immobilization on the interface of recombinant virus-specific polymer-based nano- and microparticles. It is assumed that protein-particle conjugates could be able to bind virions followed by engulfment by immune cells. The study was aimed to examine the effect of polylactic acid (PLA) and PLA block-copolymer with polyethylene glycol (PLA-PEG)-based micro- and nanoparticles on the cellular immune response against polymeric particle-bound antigen. Materials and methods. A recombinant chimeric protein beta-2-microglobulin — green fluorescent protein (β2M-sfGFP) was obtained by affine chromatography. The recombinant protein was immobilized onto the polymer particles, which were further used for mice immunization. Female F1 hybrid mice (CBA x C57BL) in experimental and control groups consisted of 4–6-month-old 15 animals (weighted 20–25 g). Intracellular cytokine staining was used to evaluate the cellular immune response. Results and discussion. It was shown that the nanoparticles of PLA block-copolymer with polyethylene glycol (PLA-PEG) were able to bind 10 microgram protein per 1 mg polymer. The polylactic acid nanoparticles were able to bind 2,3 microgram protein per 1 mg polymer. In experiment, mice in group 1 were immunized with 100 nm PLA-PEG particle-β2M-sfGFP conjugate, in group 2 — with same particles together with soluble β2M-sfGFP. In group 3, mice were immunized with 1400 nm PLA particles-β2M-sfGFP conjugate, and in group 4 — with same particles together with soluble protein. The spleens isolated 2 weeks after the four-time intraperitoneal immunization. Comparison of immune response between groups was assessed by nonparametric Kruskal–Wallis criterion with Tukey correction. It was shown that the number of antigen-specific CD4+ T cells produced to model protein was significantly higher after immunization with particle-β2M-sfGFP conjugate, as compared to control groups, wherein immunization was performed with a mixture of protein and unmodified particles (p < 0.001). It was found that the number of antigen-specific CD8+ T cells formed against β2m-sfGFP did not differ between all groups examined.

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Overkamp, Wout, Katrin Beilharz, Ruud Detert Oude Weme, Ana Solopova, Harma Karsens, Ákos T. Kovács, Jan Kok, Oscar P. Kuipers, and Jan-Willem Veening. "Benchmarking Various Green Fluorescent Protein Variants in Bacillus subtilis, Streptococcus pneumoniae, and Lactococcus lactis for Live Cell Imaging." Applied and Environmental Microbiology 79, no. 20 (August 16, 2013): 6481–90. http://dx.doi.org/10.1128/aem.02033-13.

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ABSTRACTGreen fluorescent protein (GFP) offers efficient ways of visualizing promoter activity and protein localizationin vivo, and many different variants are currently available to study bacterial cell biology. Which of these variants is best suited for a certain bacterial strain, goal, or experimental condition is not clear. Here, we have designed and constructed two “superfolder” GFPs with codon adaptation specifically forBacillus subtilisandStreptococcus pneumoniaeand have benchmarked them against five other previously available variants of GFP inB. subtilis,S. pneumoniae, andLactococcus lactis, using promoter-gfpfusions. Surprisingly, the best-performing GFP under our experimental conditions inB. subtiliswas the one codon optimized forS. pneumoniaeandvice versa. The data and tools described in this study will be useful for cell biology studies in low-GC-rich Gram-positive bacteria.

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Wang, Rongzhi, Shuangshuang Xiang, Yonghui Zhang, Qiuyu Chen, Yanfang Zhong, and Shihua Wang. "Development of a Functional Antibody by Using a Green Fluorescent Protein Frame as the Template." Applied and Environmental Microbiology 80, no. 14 (May 2, 2014): 4126–37. http://dx.doi.org/10.1128/aem.00936-14.

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ABSTRACTSingle-chain variable fragment (scFv) antibodies are widely used as diagnostic and therapeutic agents or biosensors for a majority of human disease. However, the limitations of the present scFv antibody in terms of stability, solubility, and affinity are challenging to produce by traditional antibody screening and expression formats. We describe here a feasible strategy for creating the green fluorescent protein (GFP)-based antibody. Complementarity-determining region 3 (CDR3), which retains the antigen binding activity, was introduced into the structural loops of superfolder GFP, and the result showed that CDR3-inserted GFP displayed almost the same fluorescence intensity as wild-type GFP, and the purified proteins of CDR3 insertion showed the similar binding activity to antigen as the corresponding scFv. Among of all of the CDRs, CDR3s are responsible for antigen recognition, and only the CDR3a insertion is the best format for producing GFP-based antibody binding to specific antigen. The wide versatility of this system was further verified by introducing CDR3 from other scFvs into loop 9 of GFP. We developed a feasible method for rapidly and effectively producing a high-affinity GFP-based antibody by inserting CDR3s into GFP loops. Further, the affinity can be enhanced by specific amino acids scanning and site-directed mutagenesis. Notably, this method had better versatility for creating antibodies to various antigens using GFP as the scaffold, suggesting that a GFP-based antibody with high affinity and specificity may be useful for disease diagnosis and therapy.

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Benítez-Mateos, Mehravar, Velasco-Lozano, RadmilaTomovska, Salassa, and López-Gallego. "Selective Immobilization of Fluorescent Proteins for the Fabrication of Photoactive Materials." Molecules 24, no. 15 (July 30, 2019): 2775. http://dx.doi.org/10.3390/molecules24152775.

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The immobilization of fluorescent proteins is a key technology enabling to fabricate a new generation of photoactive materials with potential technological applications. Herein we have exploited superfolder green (sGFP) and red (RFP) fluorescent proteins expressed with different polypeptide tags. We fused these fluorescent proteins to His-tags to immobilize them on graphene 3D hydrogels, and Cys-tags to immobilize them on porous microparticles activated with either epoxy or disulfide groups and with Lys-tags to immobilize them on upconverting nanoparticles functionalized with carboxylic groups. Genetically programming sGFP and RFP with Cys-tag and His-tag, respectively, allowed tuning the protein spatial organization either across the porous structure of two microbeads with different functional groups (agarose-based materials activated with metal chelates and epoxy-methacrylate materials) or across the surface of a single microbead functionalized with both metal-chelates and disulfide groups. By using different polypeptide tags, we can control the attachment chemistry but also the localization of the fluorescent proteins across the material surfaces. The resulting photoactive material formed by His-RFP immobilized on graphene hydrogels has been tested as pH indicator to measure pH changes in the alkaline region, although the immobilized fluorescent protein exhibited a narrower dynamic range to measure pH than the soluble fluorescent protein. Likewise, the immobilization of Lys-sGFP on alginate-coated upconverting nanoparticles enabled the infrared excitation of the fluorescent protein to be used as a green light emitter. These novel photoactive biomaterials open new avenues for innovative technological developments towards the fabrication of biosensors and photonic devices.

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Bishop, Alexa, Maki Kamoshita, Josiah B. Passmore, Christian Hacker, Tina A. Schrader, Hans R. Waterham, Joseph L. Costello, and Michael Schrader. "Fluorescent Tools to Analyze Peroxisome–Endoplasmic Reticulum Interactions in Mammalian Cells." Contact 2 (January 2019): 251525641984864. http://dx.doi.org/10.1177/2515256419848641.

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Peroxisomes (POs) and the endoplasmic reticulum (ER) cooperate extensively in lipid-related metabolic pathways, and the ER also provides phospholipids to enable the peroxisomal membrane to expand prior to division. Recently, we identified peroxisomal proteins, ACBD5 and ACBD4, and the ER protein vesicle-associated membrane protein-associated protein-B (VAPB) as tethering components, which physically interact to foster PO–ER associations at membrane contact sites. Overexpression or loss of these tether proteins alters the extent of PO–ER interactions, impacting on lipid exchange between these two compartments. To facilitate further studies into PO–ER associations at the level of membrane contact sites, their role, composition, and regulation, we have developed two fluorescence-based systems to monitor PO–ER interactions. We modified a proximity ligation assay and a split-fluorescence reporter system using split superfolder green fluorescent protein. Using the proximity ligation assay, we were able to measure the changes in PO–ER interactions while the split-fluorescence reporter was more limited and only allowed us to label PO–ER contacts. We show that both techniques can be useful additions to the toolkit of methods to study PO–ER associations and explore the relative merits of each.

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Liu, Min, Bin Wang, Fei Wang, Zhi Yang, Dan Gao, Chenyao Zhang, Lixin Ma, and Xiaolan Yu. "Soluble expression of single-chain variable fragment (scFv) in Escherichia coli using superfolder green fluorescent protein as fusion partner." Applied Microbiology and Biotechnology 103, no. 15 (June 7, 2019): 6071–79. http://dx.doi.org/10.1007/s00253-019-09925-6.

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Cava, Felipe, Miguel Angel de Pedro, Emilio Blas-Galindo, Geoffrey S. Waldo, Lars F. Westblade, and José Berenguer. "Expression and use of superfolder green fluorescent protein at high temperatures in vivo: a tool to study extreme thermophile biology." Environmental Microbiology 10, no. 3 (March 2008): 605–13. http://dx.doi.org/10.1111/j.1462-2920.2007.01482.x.

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Beilharz, Katrin, Renske van Raaphorst, Morten Kjos, and Jan-Willem Veening. "Red Fluorescent Proteins for Gene Expression and Protein Localization Studies in Streptococcus pneumoniae and Efficient Transformation with DNA Assembled via the Gibson Assembly Method." Applied and Environmental Microbiology 81, no. 20 (August 7, 2015): 7244–52. http://dx.doi.org/10.1128/aem.02033-15.

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ABSTRACTDuring the last decades, a wide range of fluorescent proteins (FPs) have been developed and improved. This has had a great impact on the possibilities in biological imaging and the investigation of cellular processes at the single-cell level. Recently, we have benchmarked a set of green fluorescent proteins (GFPs) and generated a codon-optimized superfolder GFP for efficient use in the important human pathogenStreptococcus pneumoniaeand other low-GC Gram-positive bacteria. In the present work, we constructed and compared four red fluorescent proteins (RFPs) inS. pneumoniae. Two orange-red variants, mOrange2 and TagRFP, and two far-red FPs, mKate2 and mCherry, were codon optimized and examined by fluorescence microscopy and plate reader assays. Notably, protein fusions of the RFPs to FtsZ were constructed by direct transformation of linear Gibson assembly (isothermal assembly) products, a method that speeds up the strain construction process significantly. Our data show that mCherry is the fastest-maturing RFP inS. pneumoniaeand is best suited for studying gene expression, while mKate2 and TagRFP are more stable and are the preferred choices for protein localization studies. The RFPs described here will be useful for cell biology studies that require multicolor labeling inS. pneumoniaeand related organisms.

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Fernandez, Juliette, Cédric Hassen-Khodja, Virginie Georget, Thierry Rose, Yves Jacob, Yves L. Janin, Sébastien Nisole, Pierre-Olivier Vidalain, and Nathalie J. Arhel. "Measuring the subcellular compartmentalization of viral infections by protein complementation assay." Proceedings of the National Academy of Sciences 118, no. 2 (January 5, 2021): e2010524118. http://dx.doi.org/10.1073/pnas.2010524118.

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The recent emergence and reemergence of viruses in the human population has highlighted the need to develop broader panels of therapeutic molecules. High-throughput screening assays opening access to untargeted steps of the viral replication cycle will provide powerful leverage to identify innovative antiviral molecules. We report here the development of an innovative protein complementation assay, termed αCentauri, to measure viral translocation between subcellular compartments. As a proof of concept, the Centauri fragment was either tethered to the nuclear pore complex or sequestered in the nucleus, while the complementary α fragment (<16 amino acids) was attached to the integrase proteins of infectious HIV-1. The translocation of viral ribonucleoproteins from the cytoplasm to the nuclear envelope or to the nucleoplasm efficiently reconstituted superfolder green fluorescent protein or NanoLuc αCentauri reporters. These fluorescence- or bioluminescence-based assays offer a robust readout of specific steps of viral infection in a multiwell format that is compatible for high-throughput screening and is validated by a short hairpin RNA-based prototype screen.

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Fleurot, Isabelle, Marina Aigle, Renaud Fleurot, Claire Darrigo, Jacques-Antoine Hennekinne, Alexandra Gruss, Elise Borezée-Durant, and Agnès Delacroix-Buchet. "Following Pathogen Development and Gene Expression in a Food Ecosystem: the Case of a Staphylococcus aureus Isolate in Cheese." Applied and Environmental Microbiology 80, no. 16 (June 13, 2014): 5106–15. http://dx.doi.org/10.1128/aem.01042-14.

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ABSTRACTHuman intoxication or infection due to bacterial food contamination constitutes an economic challenge and a public health problem. Information on thein situdistribution and expression of pathogens responsible for this risk is to date lacking, largely because of technical bottlenecks in detecting signals from minority bacterial populations within a complex microbial and physicochemical ecosystem. We simulated the contamination of a real high-risk cheese with a natural food isolate ofStaphylococcus aureus, an enterotoxin-producing pathogen responsible for food poisoning. To overcome the problem of a detection limit in a solid matrix, we chose to work with a fluorescent reporter (superfolder green fluorescent protein) that would allow spatiotemporal monitoring ofS. aureuspopulations and targeted gene expression. The combination of complementary techniques revealed thatS. aureuslocalizes preferentially on the cheese surface during ripening. Immunochemistry and confocal laser scanning microscopy enabled us to visualize, in a single image, dairy bacteria and pathogen populations, virulence gene expression, and the toxin produced. This procedure is readily applicable to other genes of interest, other bacteria, and different types of food matrices.

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Kim, Hyeran, Seul Gee Hwang, Kyeonghye Guk, Yoonji Bae, Hwangseo Park, Eun-Kyung Lim, Taejoon Kang, and Juyeon Jung. "Development of antibody against drug-resistant respiratory syncytial virus: Rapid detection of mutant virus using split superfolder green fluorescent protein-antibody system." Biosensors and Bioelectronics 194 (December 2021): 113593. http://dx.doi.org/10.1016/j.bios.2021.113593.

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Kjos, Morten, Rieza Aprianto, Vitor E. Fernandes, Peter W. Andrew, Jos A. G. van Strijp, Reindert Nijland, and Jan-Willem Veening. "Bright Fluorescent Streptococcus pneumoniae for Live-Cell Imaging of Host-Pathogen Interactions." Journal of Bacteriology 197, no. 5 (December 15, 2014): 807–18. http://dx.doi.org/10.1128/jb.02221-14.

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Streptococcus pneumoniaeis a common nasopharyngeal resident in healthy people but, at the same time, one of the major causes of infectious diseases such as pneumonia, meningitis, and sepsis. The shift from commensal to pathogen and its interaction with host cells are poorly understood. One of the major limitations for research on pneumococcal-host interactions is the lack of suitable tools for live-cell imaging. To address this issue, we developed a generally applicable strategy to create genetically stable, highly fluorescent bacteria. Our strategy relies on fusing superfolder green fluorescent protein (GFP) or a far-red fluorescent protein (RFP) to the abundant histone-like protein HlpA. Due to efficient translation and limited cellular diffusion of these fusions, the cells are 25-fold brighter than those of the currently best available imagingS. pneumoniaestrain. These novel bright pneumococcal strains are fully virulent, and the GFP reporter can be used forin situimaging in mouse tissue. We used our reporter strains to study the effect of the polysaccharide capsule, a major pneumococcal virulence factor, on different stages of infection. By dual-color live-cell imaging experiments, we show that unencapsulated pneumococci adhere significantly better to human lung epithelial cells than encapsulated strains, in line with previous data obtained by classical approaches. We also confirm with live-cell imaging that the capsule protects pneumococci from neutrophil phagocytosis, demonstrating the versatility and usability of our reporters. The described imaging tools will pave the way for live-cell imaging of pneumococcal infection and help further understanding of the mechanisms of pneumococcal pathogenesis.

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Shields, R. C., J. R. Kaspar, K. Lee, S. A. M. Underhill, and R. A. Burne. "Fluorescence Tools Adapted for Real-Time Monitoring of the Behaviors ofStreptococcusSpecies." Applied and Environmental Microbiology 85, no. 15 (May 17, 2019). http://dx.doi.org/10.1128/aem.00620-19.

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ABSTRACTTagging of bacteria with fluorescent proteins has become an essential component of modern microbiology. Fluorescent proteins can be used to monitor gene expression and biofilm growth and to visualize host-pathogen interactions. Here, we developed a collection of fluorescent protein reporter plasmids forStreptococcus mutansUA159 and other oral streptococci. Using superfolder green fluorescent protein (sfGFP) as a reporter for transcriptional activity, we were able to characterize four strong constitutive promoters inS. mutans. These promoter-sfgfpfusions worked both for single-copy chromosomal integration and on a multicopy plasmid, with the latter being segregationally stable in the absence of selective pressure under the conditions tested. We successfully labeledS. mutansUA159,Streptococcus gordoniiDL1, andStreptococcussp. strain A12 with sfGFP, DsRed-Express2 (red), and citrine (yellow). To test these plasmids under more challenging conditions, we performed mixed-species biofilm experiments and separated fluorescent populations using fluorescence-activated cell sorting (FACS). This allowed us to visualize two streptococci at a time and quantify the amounts of each species simultaneously. These fluorescent reporter plasmids add to the genetic toolbox available for the study of oral streptococci.IMPORTANCEOral streptococci are the most abundant bacteria in the mouth and have a major influence on oral health and disease. In this study, we designed and optimized the expression of fluorescent proteins inStreptococcus mutansand other oral streptococci. We monitored the levels of expression and noise (the variability in fluorescence across the population). We then created several fluorescent protein delivery systems (green, yellow, and red) for use in oral streptococci. The data show that we can monitor bacterial growth and interactionsin situ, differentiating between different bacteria growing in biofilms, the natural state of the organisms in the human mouth. These new tools will allow researchers to study these bacteria in novel ways to create more effective diagnostic and therapeutic tools for ubiquitous infectious diseases.

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Miki, Takayuki, Taichi Nakai, Masahiro Hashimoto, Keigo Kajiwara, Hiroshi Tsutsumi, and Hisakazu Mihara. "Intracellular artificial supramolecules based on de novo designed Y15 peptides." Nature Communications 12, no. 1 (June 7, 2021). http://dx.doi.org/10.1038/s41467-021-23794-6.

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AbstractDe novo designed self-assembling peptides (SAPs) are promising building blocks of supramolecular biomaterials, which can fulfill a wide range of applications, such as scaffolds for tissue culture, three-dimensional cell culture, and vaccine adjuvants. Nevertheless, the use of SAPs in intracellular spaces has mostly been unexplored. Here, we report a self-assembling peptide, Y15 (YEYKYEYKYEYKYEY), which readily forms β-sheet structures to facilitate bottom-up synthesis of functional protein assemblies in living cells. Superfolder green fluorescent protein (sfGFP) fused to Y15 assembles into fibrils and is observed as fluorescent puncta in mammalian cells. Y15 self-assembly is validated by fluorescence anisotropy and pull-down assays. By using the Y15 platform, we demonstrate intracellular reconstitution of Nck assembly, a Src-homology 2 and 3 domain-containing adaptor protein. The artificial clusters of Nck induce N-WASP (neural Wiskott-Aldrich syndrome protein)-mediated actin polymerization, and the functional importance of Nck domain valency and density is evaluated.

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Kulkarni, Surashree S., Joseph J. Johnston, Yongtao Zhu, Zachary T. Hying, and Mark J. McBride. "The Carboxy-Terminal Region ofFlavobacterium johnsoniaeSprB Facilitates Its Secretion by the Type IX Secretion System and Propulsion by the Gliding Motility Machinery." Journal of Bacteriology 201, no. 19 (July 1, 2019). http://dx.doi.org/10.1128/jb.00218-19.

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ABSTRACTFlavobacterium johnsoniaeSprB moves rapidly along the cell surface, resulting in gliding motility. SprB secretion requires the type IX secretion system (T9SS). Proteins secreted by the T9SS typically have conserved C-terminal domains (CTDs) belonging to the type A CTD or type B CTD family. Attachment of 70- to 100-amino-acid type A CTDs to a foreign protein allows its secretion. Type B CTDs are common but have received little attention. Secretion of the foreign protein superfolder green fluorescent protein (sfGFP) fused to regions spanning the SprB type B CTD (sfGFP-CTDSprB) was analyzed. CTDs of 218 amino acids or longer resulted in secretion of sfGFP, whereas a 149-amino-acid region did not. Some sfGFP was secreted in soluble form, whereas the rest was attached on the cell surface. Surface-attached sfGFP was rapidly propelled along the cell, suggesting productive interaction with the motility machinery. This did not result in rapid cell movement, which apparently requires additional regions of SprB. Secretion of sfGFP-CTDSprBrequired coexpression withsprF, which lies downstream ofsprB. SprF is similar in sequence toPorphyromonas gingivalisPorP. MostF. johnsoniaegenes encoding proteins with type B CTDs lie immediately upstream ofporP/sprF-like genes. sfGFP was fused to the type B CTD from one such protein (Fjoh_3952). This resulted in secretion of sfGFP only when it was coexpressed with its cognate PorP/SprF-like protein. These results highlight the need for extended regions of type B CTDs and for coexpression with the appropriate PorP/SprF-like protein for efficient secretion and cell surface localization of cargo proteins.IMPORTANCETheF. johnsoniaegliding motility adhesin SprB is delivered to the cell surface by the type IX secretion system (T9SS) and is rapidly propelled along the cell by the motility machinery. How this 6,497-amino-acid protein interacts with the secretion and motility machines is not known. Fusion of the C-terminal 218 amino acids of SprB to a foreign cargo protein resulted in its secretion, attachment to the cell surface, and rapid movement by the motility machinery. Efficient secretion of SprB required coexpression with the outer membrane protein SprF. Secreted proteins that have sequence similarity to SprB in their C-terminal regions are common in the phylumBacteroidetesand may have roles in adhesion, motility, and virulence.

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Kulkarni, Surashree S., Yongtao Zhu, Colton J. Brendel, and Mark J. McBride. "Diverse C-Terminal Sequences Involved in Flavobacterium johnsoniae Protein Secretion." Journal of Bacteriology 199, no. 12 (April 10, 2017). http://dx.doi.org/10.1128/jb.00884-16.

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ABSTRACT Flavobacterium johnsoniae and many related bacteria secrete proteins across the outer membrane using the type IX secretion system (T9SS). Proteins secreted by T9SSs have amino-terminal signal peptides for export across the cytoplasmic membrane by the Sec system and carboxy-terminal domains (CTDs) targeting them for secretion across the outer membrane by the T9SS. Most but not all T9SS CTDs belong to the family TIGR04183 (type A CTDs). We functionally characterized diverse CTDs for secretion by the F. johnsoniae T9SS. Attachment of the CTDs from F. johnsoniae RemA, AmyB, and ChiA to the foreign superfolder green fluorescent protein (sfGFP) that had a signal peptide at the amino terminus resulted in secretion across the outer membrane. In each case, approximately 80 to 100 amino acids from the extreme carboxy termini were needed for efficient secretion. Several type A CTDs from distantly related members of the phylum Bacteroidetes functioned in F. johnsoniae, supporting the secretion of sfGFP by the F. johnsoniae T9SS. F. johnsoniae SprB requires the T9SS for secretion but lacks a type A CTD. It has a conserved C-terminal domain belonging to the family TIGR04131, which we refer to as a type B CTD. The CTD of SprB was required for its secretion, but attachment of C-terminal regions of SprB of up to 1,182 amino acids to sfGFP failed to result in secretion. Additional features outside the C-terminal region of SprB may be required for its secretion. IMPORTANCE Type IX protein secretion systems (T9SSs) are common in but limited to members of the phylum Bacteroidetes. Most proteins that are secreted by T9SSs have conserved carboxy-terminal domains that belong to the protein domain family TIGR04183 (type A CTDs) or TIGR04131 (type B CTDs). Here, we identify features of T9SS CTDs of F. johnsoniae that are required for protein secretion and demonstrate that type A CTDs from distantly related members of the phylum function with the F. johnsoniae T9SS to secrete the foreign protein sfGFP. In contrast, type B CTDs failed to target sfGFP for secretion, suggesting a more complex association with the T9SS.

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Inaba, Yuta, Indrani Banerjee, Timothy Kernan, and Scott Banta. "Transposase-Mediated Chromosomal Integration of Exogenous Genes inAcidithiobacillus ferrooxidans." Applied and Environmental Microbiology 84, no. 21 (August 24, 2018). http://dx.doi.org/10.1128/aem.01381-18.

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ABSTRACTThe development ofAcidithiobacillus ferrooxidansas a non-model host organism for synthetic biology is hampered by a lack of genetic tools and techniques. New plating and liquid-based selection methods were developed to improve the identification of transformed cell lines. Enabled by these methods, a hyperactive transposase was used to generate mutants with integrated genes for the expression of the superfolder green fluorescent protein (sfGFP) gene or a 2-keto decarboxylase (KDC) gene, which enabled the production and secretion of isobutyric acid (IBA). An inverse PCR method was used to identify the insertion sites of the KDC gene in several mutants, leading to the identification of a region on the chromosome that may be suitable for future genetic insertions. These results demonstrate that functional exogenous metabolic genes have been chromosomally integrated intoA. ferrooxidans, and this advance will facilitate the future development of these cells for new biotechnology applications.IMPORTANCEAcidithiobacillus ferrooxidansis an iron- and sulfur-oxidizing chemolithoautotroph and is a key member of the microbial consortia used in industrial biomining applications. There is interest in exploiting these cells for other metal recovery applications as well as in developing them as unique nonmodel microbial cell factories. Plasmid-driven expression of exogenous genes has been reported, and homologous recombination has been used to knock out some gene expression. Here, new selection protocols facilitated the development of a transposition method for chromosomal integration of exogenous genes intoA. ferrooxidans. This greatly expands the available genetic toolbox, which will open the door to greater metabolic engineering efforts for these cells.

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Godeux, Anne-Sophie, Agnese Lupo, Marisa Haenni, Simon Guette-Marquet, Gottfried Wilharm, Maria-Halima Laaberki, and Xavier Charpentier. "Fluorescence-Based Detection of Natural Transformation in Drug-ResistantAcinetobacter baumannii." Journal of Bacteriology 200, no. 19 (July 16, 2018). http://dx.doi.org/10.1128/jb.00181-18.

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ABSTRACTAcinetobacter baumanniiis a nosocomial agent with a high propensity for developing resistance to antibiotics. This ability relies on horizontal gene transfer mechanisms occurring in theAcinetobactergenus, including natural transformation. To study natural transformation in bacteria, the most prevalent method uses selection for the acquisition of an antibiotic resistance marker in a target chromosomal locus by the recipient cell. Most clinical isolates ofA. baumanniiare resistant to multiple antibiotics, limiting the use of such selection-based methods. Here, we report the development of a phenotypic and selection-free method based on flow cytometry to detect transformation events in multidrug-resistant (MDR) clinicalA. baumanniiisolates. To this end, we engineered a translational fusion between the abundant and conservedA. baumanniinucleoprotein (HU) and the superfolder green fluorescent protein (sfGFP). The new method was benchmarked against the conventional antibiotic selection-based method. Using this new method, we investigated several parameters affecting transformation efficiencies and identified conditions of transformability one hundred times higher than those previously reported. Using optimized transformation conditions, we probed natural transformation in a set of MDR clinical and nonclinical animalA. baumanniiisolates. Regardless of their origin, the majority of the isolates displayed natural transformability, indicative of a conserved trait in the species. Overall, this new method and optimized protocol will greatly facilitate the study of natural transformation in the opportunistic pathogenA. baumannii.IMPORTANCEAntibiotic resistance is a pressing global health concern with the rise of multiple and panresistant pathogens. The rapid and unfailing resistance to multiple antibiotics of the nosocomial agentAcinetobacter baumannii, notably to carbapenems, prompt to understand the mechanisms behind acquisition of new antibiotic resistance genes. Natural transformation, one of the horizontal gene transfer mechanisms in bacteria, was only recently described inA. baumanniiand could explain its ability to acquire resistance genes. We developed a reliable method to probe and study natural transformation mechanism inA. baumannii. More broadly, this new method based on flow cytometry will allow experimental detection and quantification of horizontal gene transfer events in multidrug-resistantA. baumannii.

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Goudeau, Jérôme, Catherine S. Sharp, Jonathan Paw, Laura Savy, Manuel D. Leonetti, Andrew G. York, Dustin L. Updike, Cynthia Kenyon, and Maria Ingaramo. "Split-wrmScarlet and split-sfGFP: tools for faster, easier fluorescent labeling of endogenous proteins in Caenorhabditis elegans." Genetics 217, no. 4 (February 2, 2021). http://dx.doi.org/10.1093/genetics/iyab014.

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Abstract We create and share a new red fluorophore, along with a set of strains, reagents and protocols, to make it faster and easier to label endogenous Caenorhabditis elegans proteins with fluorescent tags. CRISPR-mediated fluorescent labeling of C. elegans proteins is an invaluable tool, but it is much more difficult to insert fluorophore-size DNA segments than it is to make small gene edits. In principle, high-affinity asymmetrically split fluorescent proteins solve this problem in C. elegans: the small fragment can quickly and easily be fused to almost any protein of interest, and can be detected wherever the large fragment is expressed and complemented. However, there is currently only one available strain stably expressing the large fragment of a split fluorescent protein, restricting this solution to a single tissue (the germline) in the highly autofluorescent green channel. No available C. elegans lines express unbound large fragments of split red fluorescent proteins, and even state-of-the-art split red fluorescent proteins are dim compared to the canonical split-sfGFP protein. In this study, we engineer a bright, high-affinity new split red fluorophore, split-wrmScarlet. We generate transgenic C. elegans lines to allow easy single-color labeling in muscle or germline cells and dual-color labeling in somatic cells. We also describe a novel expression strategy for the germline, where traditional expression strategies struggle. We validate these strains by targeting split-wrmScarlet to several genes whose products label distinct organelles, and we provide a protocol for easy, cloning-free CRISPR/Cas9 editing. As the collection of split-FP strains for labeling in different tissues or organelles expands, we will post updates at doi.org/10.5281/zenodo.3993663

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Ernst, Patrick, Andreas Plückthun, and Peer R. E. Mittl. "Structural analysis of biological targets by host:guest crystal lattice engineering." Scientific Reports 9, no. 1 (October 23, 2019). http://dx.doi.org/10.1038/s41598-019-51017-y.

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Abstract To overcome the laborious identification of crystallisation conditions for protein X-ray crystallography, we developed a method where the examined protein is immobilised as a guest molecule in a universal host lattice. We applied crystal engineering to create a generic crystalline host lattice under reproducible, predefined conditions and analysed the structures of target guest molecules of different size, namely two 15-mer peptides and green fluorescent protein (sfGFP). A fusion protein with an N-terminal endo-α-N-acetylgalactosaminidase (EngBF) domain and a C-terminal designed ankyrin repeat protein (DARPin) domain establishes the crystal lattice. The target is recruited into the host lattice, always in the same crystal form, through binding to the DARPin. The target structures can be determined rapidly from difference Fourier maps, whose quality depends on the size of the target and the orientation of the DARPin.

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Liang, Tianxin, Jun Sun, Shuyun Ju, Shenyi Su, Lirong Yang, and Jianping Wu. "Construction of T7-Like Expression System in Pseudomonas putida KT2440 to Enhance the Heterologous Expression Level." Frontiers in Chemistry 9 (July 16, 2021). http://dx.doi.org/10.3389/fchem.2021.664967.

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Pseudomonas putida KT2440 has become an attractive chassis for heterologous expression with the development of effective genetic manipulation tools. Improving the level of transcriptional regulation is particularly important for extending the potential of P. putida KT2440 in heterologous expression. Although many strategies have been applied to enhance the heterologous expression level in P. putida KT2440, it was still at a relatively low level. Herein we constructed a T7-like expression system in P. putida KT2440, mimicking the pET expression system in Escherichia coli, which consisted of T7-like RNA polymerase (MmP1) integrated strain and the corresponding expression vector for the heterologous expression enhancement. With the optimization of the insertion site and the copy number of RNA polymerase (RNAP), the relative fluorescence intensity (RFI) of the super-folder green fluorescent protein (sfGFP) was improved by 1.4-fold in MmP1 RNAP integrated strain. The induction point and IPTG concentration were also optimized. This strategy was extended to the gene-reduced strain EM42 and the expression of sfGFP was improved by 2.1-fold. The optimal RNAP integration site was also used for introducing T7 RNAP in P. putida KT2440 and the expression level was enhanced, indicating the generality of the integration site for the T7 expression system. Compared to other inducible expression systems in KT2440, the heterologous expression level of the Mmp1 system and T7 system were more than 2.5 times higher. Furthermore, the 3.6-fold enhanced expression level of a difficult-to-express nicotinate dehydrogenase from Comamonas testosteroni JA1 verified the efficiency of the T7-like expression system in P. putida KT2440. Taken together, we constructed and optimized the T7-like and T7 expression system in P. putida, thus providing a set of applicable chassis and corresponding plasmids to improve recombinant expression level, expecting to be used for difficult-to-express proteins.

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Attia, Mohamed A., and Harry Brumer. "Characterization of a galactosyl-binding protein module from a Cellvibrio japonicus endo-xyloglucanase defines a new family of Carbohydrate Binding Modules." Applied and Environmental Microbiology, December 18, 2020, AEM.02634–20. http://dx.doi.org/10.1128/aem.02634-20.

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Carbohydrate-binding modules (CBMs) are usually appended to carbohydrate-active enzymes (CAZymes) and serve to potentiate catalytic activity, e.g. by increasing substrate affinity. The Gram-negative soil saprophyte Cellvibrio japonicus is valuable source for CAZyme and CBM discovery and characterization, due to its innate ability to degrade a wide array of plant polysaccharides. Bioinformatic analysis of the CJA_2959 gene product from C. japonicus revealed a modular architecture consisting of a fibronectin type III (Fn3) module, a cryptic module of unknown function (“X181”), and a Glycoside Hydrolase Family 5 subfamily 4 (GH5_4) catalytic module. We previously demonstrated that the last of these, CjGH5F, is an efficient and specific endo-xyloglucanase [Attia et al. 2018. Biotechnol. Biofuels, 11: 45]. In the present study, C-terminal fusion of superfolder green fluorescent protein in tandem with the Fn3-X181 modules enabled recombinant production and purification from Escherichia coli. Native affinity gel electrophoresis revealed binding specificity for the terminal galactose-containing plant polysaccharides galactoxyloglucan and galactomannan. Isothermal titration calorimetry further evidenced a preference for galactoxyloglucan polysaccharide over short oligosaccharides comprising the limit-digest product of CjGH5F. Thus, our results identify the X181 module as the defining member of a new CBM family, CBM88. In addition to directly revealing the function of this CBM in the context of xyloglucan metabolism by C. japonicus, this study will guide future bioinformatic and functional analyses across microbial (meta)genomes.Importance This study reveals Carbohydrate Binding Module Family 88 (CBM88) as a new family of galactose-binding protein modules, which are found in series with diverse microbial glycoside hydrolases, polysaccharide lyases, and carbohydrate esterases. The definition of CBM88 in the Carbohydrate-Active Enzymes classification (http://www.cazy.org/CBM88.html) will significantly enable future microbial (meta)genome analysis and functional studies.

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Söderberg, Jenny Johansson, Miriam Grgic, Erik Hjerde, and Peik Haugen. "Aliivibrio wodanis as a production host: development of genetic tools for expression of cold-active enzymes." Microbial Cell Factories 18, no. 1 (November 11, 2019). http://dx.doi.org/10.1186/s12934-019-1247-1.

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Abstract Background Heterologous production of cold-adapted proteins currently represents one of the greatest bottlenecks in the ongoing bioprospecting efforts to find new enzymes from low-temperature environments, such as, the polar oceans that represent essentially untapped resources in this respect. In mesophilic expression hosts such as Escherichia coli, cold-adapted enzymes often form inactive aggregates. Therefore it is necessary to develop new low-temperature expression systems, including identification of new host organisms and complementary genetic tools. Psychrophilic bacteria, including Pseudoalteromonas haloplanktis, Shewanella and Rhodococcus erythropolis have all been explored as candidates for such applications. However to date none of these have found widespread use as efficient expression systems, or are commercially available. In the present work we explored the use of the sub-Arctic bacterium Aliivibrio wodanis as a potential host for heterologous expression of cold-active enzymes. Results We tested 12 bacterial strains, as well as available vectors, promoters and reporter systems. We used RNA-sequencing to determine the most highly expressed genes and their intrinsic promoters in A. wodanis. In addition we examined a novel 5′-fusion to stimulate protein production and solubility. Finally we tested production of a set of “difficult-to-produce” enzymes originating from various bacteria and one Archaea. Our results show that cold-adapted enzymes can be produced in soluble and active form, even in cases when protein production failed in E. coli due to the formation of inclusion bodies. Moreover, we identified a 60-bp/20-aa fragment from the 5′-end of the AW0309160_00174 gene that stimulates expression of Green Fluorescent Protein and improves production of cold-active enzymes when used as a 5′-fusion. A 25-aa peptide from the same protein enhanced secretion of a 25-aa-sfGFP fusion. Conclusions Our results indicate the use of A. wodanis and associated genetic tools for low-temperature protein production and indicate that A. wodanis represents an interesting platform for further development of a protein production system that can promote further cold-enzyme discoveries.

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Moradali, M. Fata, Shirin Ghods, and Bernd H. A. Rehm. "Activation Mechanism and Cellular Localization of Membrane-Anchored Alginate Polymerase in Pseudomonas aeruginosa." Applied and Environmental Microbiology 83, no. 9 (March 3, 2017). http://dx.doi.org/10.1128/aem.03499-16.

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ABSTRACT The exopolysaccharide alginate, produced by the opportunistic human pathogen Pseudomonas aeruginosa, confers a survival advantage to the bacterium by contributing to the formation of characteristic biofilms during infection. Membrane-anchored proteins Alg8 (catalytic subunit) and Alg44 (copolymerase) constitute the alginate polymerase that is being activated by the second messenger molecule bis-(3′, 5′)-cyclic dimeric GMP (c-di-GMP), but the mechanism of activation remains elusive. To shed light on the c-di-GMP-mediated activation of alginate polymerization in vivo, an in silico structural model of Alg8 fused to the c-di-GMP binding PilZ domain informed by the structure of cellulose synthase, BcsA, was developed. This structural model was probed by site-specific mutagenesis and different cellular levels of c-di-GMP. Results suggested that c-di-GMP-mediated activation of alginate polymerization involves amino acids residing at two loops, including H323 (loop A) and T457 and E460 (loop B), surrounding the catalytic site in the predicted model. The activities of the respective Alg8 variants suggested that c-di-GMP-mediated control of substrate access to the catalytic site of Alg8 is dissimilar to the known activation mechanism of BcsA. Alg8 variants responded differently to various c-di-GMP levels, while MucR imparted c-di-GMP for activation of alginate polymerase. Furthermore, we showed that Alg44 copolymerase constituted a stable dimer, with its periplasmic domains required for protein localization and alginate polymerization and modification. Superfolder green fluorescent protein (GFP) fusions of Alg8 and Alg44 showed a nonuniform, punctate, and patchy arrangement of both proteins surrounding the cell. Overall, this study provides insights into the c-di-GMP-mediated activation of alginate polymerization while assigning functional roles to Alg8 and Alg44, including their subcellular localization and distribution. IMPORTANCE The exopolysaccharide alginate is an important biofilm component of the opportunistic human pathogen P. aeruginosa and the principal cause of the mucoid phenotype that is the hallmark of chronic infections of cystic fibrosis patients. The production of alginate is mediated by interacting membrane proteins Alg8 and Alg44, while their activity is posttranslationally regulated by the second messenger c-di-GMP, a well-known regulator of the synthesis of a range of other exopolysaccharides in bacteria. This study provides new insights into the unknown activation mechanism of alginate polymerization by c-di-GMP. Experimental evidence that the activation of alginate polymerization requires the engagement of specific amino acid residues residing at the catalytic domain of Alg8 glycosyltransferase was obtained, and these residues are proposed to exert an allosteric effect on the PilZAlg44 domain upon c-di-GMP binding. This mechanism is dissimilar to the proposed mechanism of the autoinhibition of cellulose polymerization imposed by salt bridge formation between amino acid residues and released upon c-di-GMP binding, leading to activation of polymerization. On the other hand, conserved amino acid residues in the periplasmic domain of Alg44 were found to be involved in alginate polymerization as well as modification events, i.e., acetylation and epimerization. Due to the critical role of c-di-GMP in the regulation of many biological processes, particularly the motility-sessility switch and also the emergence of persisting mucoid phenotypes, these results aid to reach a better understanding of biofilm-associated regulatory networks and c-di-GMP signaling and might assist the development of inhibitory drugs.

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Journal articles on the topic 'SfGFP, superfolder green fluorescent protein'

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