Journal articles: 'Maltose binding protein (MBP)'

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Morón, Carlos, Enrique Tremps, Alfonso Garcia, and Jose Andrés Somolinos. "Development of an Electrochemical Maltose Biosensor." Key Engineering Materials 495 (November 2011): 116–19. http://dx.doi.org/10.4028/www.scientific.net/kem.495.116.

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In this work, electrochemical maltose biosensors based on mutants of the maltose binding protein (MBP) are developed. A rutheniumIIcomplex (RuII), which is covalently attached to MBP, serves as an electrochemical reporter of MBP conformational changes. Biosensors were made through direct attachment of RuIIcomplex modified MBP to gold electrode surfaces. The responses of some individual mutants were evaluated using square wave voltammetry. A maltose-dependent change in Faradic current and capacitance was observed. It is therefore demonstrated that biosensors using generically this family of bacterial periplasmic binding proteins (bPBP) can be made lending themselves to facile biorecognition element preparation and low cost electrochemical transduction.

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Reich-Slotky, Ronit, Cynthia Panagiotidis, Moraima Reyes, and Howard A. Shuman. "The Detergent-Soluble Maltose Transporter Is Activated by Maltose Binding Protein and Verapamil." Journal of Bacteriology 182, no. 4 (February 15, 2000): 993–1000. http://dx.doi.org/10.1128/jb.182.4.993-1000.2000.

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ABSTRACT The maltose transporter FGK2 complex of Escherichia coli was purified with the aid of a glutathioneS-transferase molecular tag. In contrast to the membrane-associated form of the complex, which requires liganded maltose binding protein (MBP) for ATPase activity, the purified detergent-soluble complex exhibited a very high level of ATPase activity. This uncoupled activity was not due to dissociation of the MalK ATPase subunit from the integral membrane protein MalF and MalG subunits. The detergent-soluble ATPase activity of the complex could be further stimulated by wild-type MBP but not by a signaling-defective mutant MBP. Wild-type MBP increased the V max of the ATPase 2.7-fold but had no effect on the Km of the enzyme for ATP. When the detergent-soluble complex was reconstituted in proteoliposomes, it returned to being dependent on MBP for activation of ATPase, consistent with the idea that the structural changes induced in the complex by detergent that result in activation of the ATPase are reversible. The uncoupled ATPase activity resembled the membrane-bound activity of the complex also with respect to sensitivity to NaN3, as well as a mercurial,p-chloromercuribenzosulfonic acid. Verapamil, a compound that activates the ATPase activity of the multiple drug resistance P-glycoprotein, activated the maltose transporter ATPase as well. The activation of this bacterial transporter by verapamil suggests that a structural feature that is conserved among both eukaryotic and prokaryotic ATP binding cassette transporters is responsible for this activation.

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Braitsch, Michaela, Hanspeter Kählig, Georg Kontaxis, Michael Fischer, Toshinari Kawada, Robert Konrat, and Walther Schmid. "Synthesis of fluorinated maltose derivatives for monitoring protein interaction by 19F NMR." Beilstein Journal of Organic Chemistry 8 (March 27, 2012): 448–55. http://dx.doi.org/10.3762/bjoc.8.51.

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A novel reporter system, which is applicable to the 19F NMR investigation of protein interactions, is presented. This approach uses 2-F-labeled maltose as a spy ligand to indirectly probe protein–ligand or protein–protein interactions of proteins fused or tagged to the maltose-binding protein (MBP). The key feature is the simultaneous NMR observation of both 19F NMR signals of gluco/manno-type-2-F-maltose-isomers; one isomer (α-gluco-type) binds to MBP and senses the protein interaction, and the nonbinding isomers (β-gluco- and/or α/β-manno-type) are utilized as internal references. Moreover, this reporter system was used for relative affinity studies of fluorinated and nonfluorinated carbohydrates to the maltose-binding protein, which were found to be in perfect agreement with published X-ray data. The results of the NMR competition experiments together with the established correlation between 19F chemical shift data and molecular interaction patterns, suggest valuable applications for studies of protein–ligand interaction interfaces.

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Williard, Alexander C., Hannah J. Switzer, Christina A. Howard, Rui Yin, Brent L. Russell, Ritwik Sanyal, Shaun Yu, et al. "Protein Modification Employing Non-Canonical Amino Acids to Prepare SUMOylation Detecting Bioconjugates." Pharmaceutics 14, no. 12 (December 16, 2022): 2826. http://dx.doi.org/10.3390/pharmaceutics14122826.

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Protein modification with non-canonical amino acids (ncAAs) represents a useful technology to afford homogenous samples of bioconjugates with site-specific modification. This technique can be directly applied to the detection of aberrant SUMOylation patterns, which are often indicative of disease states. Modified SUMO-trapping proteins, consisting of a catalytically inactive ULP1 fragment (UTAG) fused to the maltose-binding protein MBP, are useful reagents for the binding and labeling of SUMOylated proteins. Mutation of this UTAG fusion protein to facilitate amber suppression technologies for the genetic incorporation of ncAAs was assessed to provide a functional handle for modification. Ultimately, two sites in the maltose-binding protein (MBP) fusion were identified as ideal for incorporation and bioconjugation without perturbation to the SUMO-trapping ability of the UTAG protein. This functionality was then employed to label SUMOylated proteins in HeLa cells and demonstrate their enrichment in the nucleus. This modified UTAG-MBP-ncAA protein has far-reaching applications for both diagnostics and therapeutics.

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Waugh, David S. "The remarkable solubility-enhancing power of Escherichia coli maltose-binding protein." Postępy Biochemii 62, no. 3 (November 15, 2016): 377–82. http://dx.doi.org/10.18388/pb.2016_41.

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A common problem encountered during the production of recombinant proteins, particularly in bacteria, is their tendency to accumulate in an insoluble and inactive form (i.e., as inclusion bodies). Although sometimes it is possible to convert the aggregated material into native, biologically active protein, this is a time-consuming, costly, and uncertain undertaking. Consequently, a general means of circumventing the formation of inclusion bodies is highly desirable. During the 1990s, it was serendipitously discovered that certain highly soluble proteins have the ability to enhance the solubility of their fusion partners, thereby preventing them from forming insoluble aggregates. In the ensuing years, Escherichia coli maltose-binding protein (MBP) has emerged as one of the most effective solubility enhancers. Moreover, once rendered soluble by fusion to MBP, many proteins are able to fold into their biologically active conformations. This brief review article focuses on our current understanding of what makes MBP such an effective solubility enhancer and how it facilitates the proper folding of its fusion partners.

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Carter, Eric L., and Robert P. Hausinger. "Characterization of the Klebsiella aerogenes Urease Accessory Protein UreD in Fusion with the Maltose Binding Protein." Journal of Bacteriology 192, no. 9 (March 5, 2010): 2294–304. http://dx.doi.org/10.1128/jb.01426-09.

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ABSTRACT Assembly of the Klebsiella aerogenes urease metallocenter requires four accessory proteins, UreD, UreE, UreF, and UreG, to effectively deliver and incorporate two Ni2+ ions into the nascent active site of the urease apoprotein (UreABC). Each accessory protein has been purified and characterized with the exception of UreD due to its insolubility when it is overproduced in recombinant cells. In this study, a translational fusion was made between the maltose binding protein (MBP) and UreD, with the resulting MBP-UreD found to be soluble in Escherichia coli cell extracts and able to complement a ΔureD-urease cluster in this host microorganism. MBP-UreD was purified as a large multimer (>670 kDa) that bound approximately 2.5 Ni2+ ions (Kd of ∼50 μM, where Kd is the dissociation constant) per UreD protomer according to equilibrium dialysis measurements. Zn2+ directly competes with 10-fold higher affinity (∼4 Zn2+ ions per protomer; Kd of 5 μM) for the Ni2+ binding sites. MBP pulldown experiments demonstrated that the UreD domain of MBP-UreD formed in vivo complexes with UreF, UreG, UreF plus UreG, or UreABC when these proteins were overproduced in the same E. coli cells. In addition, a UreABC-(MBP-UreD)-UreFG complex was observed in cells producing all urease components. Comparative in vitro binding experiments with purified proteins demonstrated an approximate 1:1 binding ratio between the UreD domain of MBP-UreD and the UreF domain of the UreEF fusion, only weak or transient interaction between MBP-UreD and UreG, and no binding with UreABC. These studies are the first to describe the properties of purified UreD, and they extend our understanding of its binding partners both in vitro and in the cell.

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Munson, George P., Lisa G. Holcomb, Heather L. Alexander, and June R. Scott. "In Vitro Identification of Rns-Regulated Genes." Journal of Bacteriology 184, no. 4 (February 15, 2002): 1196–99. http://dx.doi.org/10.1128/jb.184.4.1196-1199.2002.

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ABSTRACT To identify Rns-regulated genes, a maltose binding protein (MBP)-Rns fusion protein was used to isolate DNA fragments from enterotoxigenic Escherichia coli genomic DNA that carry Rns binding sites. In vivo transcription fusion analysis shows that Rns positively regulates the expression of the open reading frame of yiiS, which lies immediately downstream of one MBP-Rns-bound fragment.

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Li, B. W., R. Chandrashekar, and G. J. Weil. "Vaccination with recombinant filarial paramyosin induces partial immunity to Brugia malayi infection in jirds." Journal of Immunology 150, no. 5 (March 1, 1993): 1881–85. http://dx.doi.org/10.4049/jimmunol.150.5.1881.

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Abstract Vaccination with irradiated infective larvae induces partial protective immunity to infection with the filarial nematode Brugia malayi in jirds. Prior studies have shown that such immunization stimulates a much stronger antibody response to recombinant and native filarial paramyosin than that seen after normal infection. To determine whether vaccination with recombinant paramyosin could induce protective immunity to larval challenge, jirds were immunized with either B. malayi paramyosin and maltose binding protein (BM5-MBP) (fusion protein of B. malayi paramyosin and maltose-binding protein), MBP alone, or recombinant Dirofilaria immitis paramyosin. Animals were challenged with 100 infective larvae s.c. 8 wk after the second immunization. Necropsies were performed 16 wk after challenge. Vaccination with BM5-MBP induced significant protective immunity; adult worm recoveries, worm lengths, and blood microfilaria counts were reduced in the BM5-MBP group relative to the MBP control group. The reductions in adult worm recoveries (43%) and female worm lengths (10%) were statistically significant (p < 0.05). Interestingly, protective immunity was not induced by immunization with D. immitis paramyosin. Additional studies are needed to identify mechanisms involved in protective immunity induced by BM5-MBP and to understand the differential activity of the two closely related recombinant paramyosin proteins in this model.

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THOMAS, Stephen, Salvador SORIANO, Clive d'SANTOS, and George BANTING. "Expression of recombinant rat myo-inositol 1,4,5-trisphosphate 3-kinase B suggests a regulatory role for its N-terminus." Biochemical Journal 319, no. 3 (November 1, 1996): 713–16. http://dx.doi.org/10.1042/bj3190713.

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We have expressed rat myo-inositol 1,4,5-trisphosphate (IP3) 3-kinase B as both a full-length, recombinant, non-fusion protein and a full-length, recombinant, fusion protein with maltose-binding protein (MBP) in Escherichia coli. The fusion protein with MBP is soluble, binds calmodulin and is enzymically active whereas the non-fusion protein is insoluble and does not bind calmodulin unless co-expressed with bacterial chaperone proteins (either GroES and GroEL, or DnaK, DnaJ and GrpE). However, soluble, calmodulin-binding non-fusion IP3 3-kinase B is enzymically inactive. The catalytic domain of the enzyme has previously been shown to reside near the C-terminus; the results we present suggest an auto-regulatory role for the N-terminus.

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Fernandez, Stefan, Dupeh R. Palmer, Monika Simmons, Peifang Sun, John Bisbing, Sasha McClain, Sachin Mani, Timothy Burgess, Vicky Gunther, and Wellington Sun. "Potential Role for Toll-Like Receptor 4 in Mediating Escherichia coli Maltose-Binding Protein Activation of Dendritic Cells." Infection and Immunity 75, no. 3 (January 12, 2007): 1359–63. http://dx.doi.org/10.1128/iai.00486-06.

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ABSTRACT The Escherichia coli maltose-binding protein (MBP) is used to increase the stability and solubility of proteins in bacterial protein expression systems and is increasingly being used to facilitate the production and delivery of subunit vaccines against various pathogenic bacteria and viruses. The MBP tag is presumed inert, with minimum effects on the bioactivity of the tagged protein or its biodistribution. However, few studies have characterized the immunological attributes of MBP. Here, we analyze the phenotypic and functional outcomes of MBP-treated dendritic cells (DCs) and show that MBP induces DC activation and production of proinflammatory cytokines (interleukin-1β [IL-1β], IL-6, IL-8, tumor necrosis factor alpha, and IL-12p70) within 24 h and strongly increases Iκβ phosphorylation in treated cells. Interestingly, phosphorylation of Iκβ was largely abrogated by the addition of anti-human Toll-like receptor 4 (TLR4) antibodies, indicating that MBP activates signaling for DC maturation via TLR4. Consistent with this hypothesis, MBP activated the TLR4-expressing cell line 293-hTLR4A but not control cultures to secrete IL-8. The observed data were independent of lipopolysaccharide contamination and support a role for TLR4 in mediating the effects of MBP. These results provide insight into a mechanism by which MBP might enhance immune responses to vaccine fusion proteins.

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Wang, Nian, Shi-En Lu, Angela R. Records, and Dennis C. Gross. "Characterization of the Transcriptional Activators SalA and SyrF, Which Are Required for Syringomycin and Syringopeptin Production by Pseudomonas syringae pv. syringae." Journal of Bacteriology 188, no. 9 (May 1, 2006): 3290–98. http://dx.doi.org/10.1128/jb.188.9.3290-3298.2006.

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ABSTRACT Production of the phytotoxins syringomycin and syringopeptin by Pseudomonas syringae pv. syringae is controlled by the regulatory genes salA and syrF. Analysis with 70-mer oligonucleotide microarrays established that the syr-syp genes responsible for synthesis and secretion of syringomycin and syringopeptin belong to the SyrF regulon. Vector pMEKm12 was successfully used to express both SalA and SyrF proteins fused to a maltose-binding protein (MBP) in Escherichia coli and P. syringae pv. syringae. Both the MBP-SalA and MBP-SyrF fusion proteins were purified by maltose affinity chromatography. Gel shift analysis revealed that the purified MBP-SyrF, but not the MBP-SalA fusion protein, bound to a 262-bp fragment of the syrB1 promoter region containing the syr-syp box. Purified MBP-SalA caused a shift of a 324-bp band containing the putative syrF promoter. Gel filtration analysis and cross-linking experiments indicated that both SalA and SyrF form homodimers in vitro. Overexpression of the N-terminal regions of SalA and SyrF resulted in decreased syringomycin production by strain B301D and reduced levels of β-glucuronidase activities of the sypA::uidA and syrB1::uidA reporters by 59% to 74%. The effect of SalA on the expression of the syr-syp genes is mediated by SyrF, which activates the syr-syp genes by directly binding to the promoter regions. Both SalA and SyrF resemble other LuxR family proteins in dimerization and interaction with promoter regions of target genes.

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Tanaka, Yoshino, Yoshihiko Nanasato, Kousei Omura, Keita Endoh, Tsuyoshi Kawano, and Takashi Iwasaki. "Direct protein delivery into intact plant cells using polyhistidine peptides." Bioscience, Biotechnology, and Biochemistry 85, no. 6 (March 31, 2021): 1405–14. http://dx.doi.org/10.1093/bbb/zbab055.

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ABSTRACT Polyhistidine peptides (PHPs), sequences comprising only histidine residues (>His8), are effective cell-penetrating peptides for plant cells. Using PHP-fusion proteins, we aimed to deliver proteins into cultured plant cells from Nicotiana tabacum, Oryza sativa, and Cryptomeria japonica. Co-cultivation of cultured cells with fusion proteins combining maltose-binding protein (MBP), red fluorescent protein (RFP), and various PHPs (MBP–RFP–His8–His20) in one polypeptide showed the cellular uptake of fusion proteins in all plant cell lines. Maximum intracellular fluorescence was shown in MBP-RFP-His20. Further, adenylate cyclase (CyaA), a synthase of cyclic adenosine monophosphate (cAMP) activated by cytosolic calmodulin, was used as a reporter for protein delivery in living cells. A fusion protein combining MBP, RFP, CyaA, and His20 (MBP–RFP–CyaA–His20) was delivered into plant cells and increased intracellular fluorescence and cAMP production in all cell lines. The present study demonstrates that PHPs are effective carriers of proteins into the intracellular space of various cultured plant cells.

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Gwack, Yousang, Hyouna Yoo, Inyoung Song, Joonho Choe, and Jang H. Han. "RNA-Stimulated ATPase and RNA Helicase Activities and RNA Binding Domain of Hepatitis G Virus Nonstructural Protein 3." Journal of Virology 73, no. 4 (April 1, 1999): 2909–15. http://dx.doi.org/10.1128/jvi.73.4.2909-2915.1999.

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ABSTRACT Hepatitis G virus (HGV) nonstructural protein 3 (NS3) contains amino acid sequence motifs typical of ATPase and RNA helicase proteins. In order to examine the RNA helicase activity of the HGV NS3 protein, the NS3 region (amino acids 904 to 1580) was fused with maltose-binding protein (MBP), and the fusion protein was expressed inEscherichia coli and purified with amylose resin and anion-exchange chromatography. The purified MBP-HGV/NS3 protein possessed RNA-stimulated ATPase and RNA helicase activities. Characterization of the ATPase and RNA helicase activities of MBP-HGV/NS3 showed that the optimal reaction conditions were similar to those of other Flaviviridae viral NS3 proteins. However, the kinetic analysis of NTPase activity showed that the MBP-HGV/NS3 protein had several unique properties compared to the otherFlaviviridae NS3 proteins. The HGV NS3 helicase unwinds RNA-RNA duplexes in a 3′-to-5′ direction and can unwind RNA-DNA heteroduplexes and DNA-DNA duplexes as well. In a gel retardation assay, the MBP-HGV/NS3 helicase bound to RNA, RNA/DNA, and DNA duplexes with 5′ and 3′ overhangs but not to blunt-ended RNA duplexes. We also found that the conserved motif VI was important for RNA binding. Further deletion mapping showed that the RNA binding domain was located between residues 1383 and 1395, QRRGRTGRGRSGR. Our data showed that the MBP-HCV/NS3 protein also contains the RNA binding domain in the similar domain.

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Reuten, Raphael, Denise Nikodemus, Maria B. Oliveira, Trushar R. Patel, Bent Brachvogel, Isabelle Breloy, Jörg Stetefeld, and Manuel Koch. "Maltose-Binding Protein (MBP), a Secretion-Enhancing Tag for Mammalian Protein Expression Systems." PLOS ONE 11, no. 3 (March 30, 2016): e0152386. http://dx.doi.org/10.1371/journal.pone.0152386.

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Gurung, Ratna B., Douglas J. Begg, Auriol C. Purdie, John P. Bannantine, and Richard J. Whittington. "Antigenicity of Recombinant Maltose Binding Protein-Mycobacterium avium subsp. paratuberculosis Fusion Proteins with and without Factor Xa Cleaving." Clinical and Vaccine Immunology 20, no. 12 (October 16, 2013): 1817–26. http://dx.doi.org/10.1128/cvi.00596-13.

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ABSTRACTMycobacterium aviumsubsp.paratuberculosiscauses Johne's disease (JD) in ruminants. Proteomic studies have shown thatM. aviumsubsp.paratuberculosisexpresses certain proteins when exposed toin vitrophysiological stress conditions similar to the conditions experienced within a host during natural infection. Such proteins are hypothesized to be expressedin vivo, are recognized by the host immune system, and may be of potential use in the diagnosis of JD. In this study, 50 recombinant maltose binding protein (MBP)-M. aviumsubsp.paratuberculosisfusion proteins were evaluated using serum samples from sheep infected withM. aviumsubsp.paratuberculosis, and 29 (58%) were found to be antigenic. Among 50 fusion proteins, 10 were evaluated in MBP fusion and factor Xa-cleaved forms. A total of 31 proteins (62%) were found to be antigenic in either MBP fusion or factor Xa-cleaved forms. Antigenicity after cleavage and removal of the MBP tag was marginally enhanced.

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Emoto, Yuri, Ryoya Katayama, Emi Hibino, Sho Ishihara, Natsuko Goda, Takeshi Tenno, Yoshihiro Kobashigawa, Hiroshi Morioka, and Hidekazu Hiroaki. "A Cost-Effective Immobilization Method for MBP Fusion Proteins on Microtiter Plates Using a Gelatinized Starch–Agarose Mixture and Its Application for Convenient Protein–Protein Interaction Analysis." Methods and Protocols 6, no. 3 (April 22, 2023): 44. http://dx.doi.org/10.3390/mps6030044.

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The detection and quantification of protein–protein interactions (PPIs) is a crucial technique that often involves the use of recombinant proteins with fusion protein tags, such as maltose-binding protein (MBP) and glutathione-S-transferase (GST). In this study, we improved the cohesive and sticky properties of gelatinized starch by supplementing it with agarose, resulting in a harder gel that could coat the bottom of a microtiter plate. The resulting gelatinized starch/agarose mixture allowed for the efficient immobilization of MBP-tagged proteins on the coated plates, enabling the use of indirect ELISA-like PPI assays. By using the enzymatic activity of GST as an indicator, we succeeded in determining the dissociation constants between MBP-tagged and GST-tagged proteins on 96-well microtiter plates and a microplate reader without any expensive specialized equipment.

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Boos, Winfried, and Howard Shuman. "Maltose/Maltodextrin System of Escherichia coli: Transport, Metabolism, and Regulation." Microbiology and Molecular Biology Reviews 62, no. 1 (March 1, 1998): 204–29. http://dx.doi.org/10.1128/mmbr.62.1.204-229.1998.

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SUMMARY The maltose system of Escherichia coli offers an unusually rich set of enzymes, transporters, and regulators as objects of study. This system is responsible for the uptake and metabolism of glucose polymers (maltodextrins), which must be a preferred class of nutrients for E. coli in both mammalian hosts and in the environment. Because the metabolism of glucose polymers must be coordinated with both the anabolic and catabolic uses of glucose and glycogen, an intricate set of regulatory mechanisms controls the expression of mal genes, the activity of the maltose transporter, and the activities of the maltose/maltodextrin catabolic enzymes. The ease of isolating many of the mal gene products has contributed greatly to the understanding of the structures and functions of several classes of proteins. Not only was the outer membrane maltoporin, LamB, or the phage lambda receptor, the first virus receptor to be isolated, but also its three-dimensional structure, together with extensive knowledge of functional sites for ligand binding as well as for phage λ binding, has led to a relatively complete description of this sugar-specific aqueous channel. The periplasmic maltose binding protein (MBP) has been studied with respect to its role in both maltose transport and maltose taxis. Again, the combination of structural and functional information has led to a significant understanding of how this soluble receptor participates in signaling the presence of sugar to the chemosensory apparatus as well as how it participates in sugar transport. The maltose transporter belongs to the ATP binding cassette family, and although its structure is not yet known at atomic resolution, there is some insight into the structures of several functional sites, including those that are involved in interactions with MBP and recognition of substrates and ATP. A particularly astonishing discovery is the direct participation of the transporter in transcriptional control of the mal regulon. The MalT protein activates transcription at all mal promoters. A subset also requires the cyclic AMP receptor protein for transcription. The MalT protein requires maltotriose and ATP as ligands for binding to a dodecanucleotide MalT box that appears in multiple copies upstream of all mal promoters. Recent data indicate that the ATP binding cassette transporter subunit MalK can directly inhibit MalT when the transporter is inactive due to the absence of substrate. Despite this wealth of knowledge, there are still basic issues that require clarification concerning the mechanism of MalT-mediated activation, repression by the transporter, biosynthesis and assembly of the outer membrane and inner membrane transporter proteins, and interrelationships between the mal enzymes and those of glucose and glycogen metabolism.

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Ye, Xiang, Leland Mayne, Zhong-yuan Kan, and S. Walter Englander. "Folding of maltose binding protein outside of and in GroEL." Proceedings of the National Academy of Sciences 115, no. 3 (January 2, 2018): 519–24. http://dx.doi.org/10.1073/pnas.1716168115.

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We used hydrogen exchange–mass spectrometry (HX MS) and fluorescence to compare the folding of maltose binding protein (MBP) in free solution and in the GroEL/ES cavity. Upon refolding, MBP initially collapses into a dynamic molten globule-like ensemble, then forms an obligatory on-pathway native-like folding intermediate (1.2 seconds) that brings together sequentially remote segments and then folds globally after a long delay (30 seconds). A single valine to glycine mutation imposes a definable folding defect, slows early intermediate formation by 20-fold, and therefore subsequent global folding by approximately twofold. Simple encapsulation within GroEL repairs the folding defect and reestablishes fast folding, with or without ATP-driven cycling. Further examination exposes the structural mechanism. The early folding intermediate is stabilized by an organized cluster of 24 hydrophobic side chains. The cluster preexists in the collapsed ensemble before the H-bond formation seen by HX MS. The V9G mutation slows folding by disrupting the preintermediate cluster. GroEL restores wild-type folding rates by restabilizing the preintermediate, perhaps by a nonspecific equilibrium compression effect within its tightly confining central cavity. These results reveal an active GroEL function other than previously proposed mechanisms, suggesting that GroEL possesses different functionalities that are able to relieve different folding problems. The discovery of the preintermediate, its mutational destabilization, and its restoration by GroEL encapsulation was made possible by the measurement of a previously unexpected type of low-level HX protection, apparently not dependent on H-bonding, that may be characteristic of proteins in confined spaces.

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Ueki, Tatsuya, Yasuhisa Sakamoto, Nobuo Yamaguchi, and Hitoshi Michibata. "Bioaccumulation of Copper Ions by Escherichia coli Expressing Vanabin Genes from the Vanadium-Rich Ascidian Ascidia sydneiensis samea." Applied and Environmental Microbiology 69, no. 11 (November 2003): 6442–46. http://dx.doi.org/10.1128/aem.69.11.6442-6446.2003.

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ABSTRACT The genes encoding two vanadium-binding proteins, vanabin1 and vanabin2, from a vanadium-rich ascidian, Ascidia sydneiensis samea, were recently identified and cloned (T. Ueki, T. Adachi, S. Kawano, M. Aoshima, N. Yamaguchi, K. Kanamori, and H. Michibata, Biochim. Biophys. Acta 1626:43-50, 2003). The vanabins were found to bind vanadium(IV), and an excess of copper(II) ions inhibited the binding of vanadium(IV) to the vanabins in vitro. In this study, we constructed Escherichia coli strains that expressed vanabin1 or vanabin2 fused to maltose-binding protein (MBP) in the periplasmic space. We found that both strains accumulated about twenty times more copper(II) ions than the control BL21 strain, while no significant accumulation of vanadium was observed. The strains expressing either MBP-vanabin1 or MBP-vanabin2 absorbed approximately 70% of the copper ions in the medium to which 10 μM copper (II) ions were initially added. The MBP-vanabin1 and MBP-vanabin2 protein expressed in the periplasm bound to copper ions at a copper:protein molar ratio of 8:1 and 5:1, respectively, but MBP did not bind to copper ions. These data showed that the metal-binding proteins vanabin1 and vanabin2 bound copper ions directly and enhanced the bioaccumulation of copper ions by E. coli.

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Posada, J., N. Yew, N. G. Ahn, G. F. Vande Woude, and J. A. Cooper. "Mos stimulates MAP kinase in Xenopus oocytes and activates a MAP kinase kinase in vitro." Molecular and Cellular Biology 13, no. 4 (April 1993): 2546–53. http://dx.doi.org/10.1128/mcb.13.4.2546-2553.1993.

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Several protein kinases, including Mos, maturation-promoting factor (MPF), mitogen-activated protein (MAP) kinase, and MAP kinase kinase (MAPKK), are activated when Xenopus oocytes enter meiosis. De novo synthesis of the Mos protein is required for progesterone-induced meiotic maturation. Recently, bacterially synthesized maltose-binding protein (MBP)-Mos fusion protein was shown to be sufficient to initiate meiosis I and MPF activation in fully grown oocytes in the absence of protein synthesis. Here we show that MAP kinase is rapidly phosphorylated and activated following injection of wild-type, but not kinase-inactive mutant, MBP-Mos into fully grown oocytes. MAP kinase activation by MBP-Mos occurs within 20 min, much more rapidly than in progesterone-treated oocytes. The MBP-Mos fusion protein also activates MPF, but MPF activation does not occur until approximately 2 h after injection. Extracts from oocytes injected with wild-type but not kinase-inactive MBP-Mos contain an activity that can phosphorylate MAP kinase, suggesting that Mos directly or indirectly activates a MAPKK. Furthermore, activated MBP-Mos fusion protein is able to phosphorylate and activate a purified, phosphatase-treated, rabbit muscle MAPKK in vitro. Thus, in oocytes, Mos is an upstream activator of MAP kinase which may function through direct phosphorylation of MAPKK.

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Posada, J., N. Yew, N. G. Ahn, G. F. Vande Woude, and J. A. Cooper. "Mos stimulates MAP kinase in Xenopus oocytes and activates a MAP kinase kinase in vitro." Molecular and Cellular Biology 13, no. 4 (April 1993): 2546–53. http://dx.doi.org/10.1128/mcb.13.4.2546.

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Several protein kinases, including Mos, maturation-promoting factor (MPF), mitogen-activated protein (MAP) kinase, and MAP kinase kinase (MAPKK), are activated when Xenopus oocytes enter meiosis. De novo synthesis of the Mos protein is required for progesterone-induced meiotic maturation. Recently, bacterially synthesized maltose-binding protein (MBP)-Mos fusion protein was shown to be sufficient to initiate meiosis I and MPF activation in fully grown oocytes in the absence of protein synthesis. Here we show that MAP kinase is rapidly phosphorylated and activated following injection of wild-type, but not kinase-inactive mutant, MBP-Mos into fully grown oocytes. MAP kinase activation by MBP-Mos occurs within 20 min, much more rapidly than in progesterone-treated oocytes. The MBP-Mos fusion protein also activates MPF, but MPF activation does not occur until approximately 2 h after injection. Extracts from oocytes injected with wild-type but not kinase-inactive MBP-Mos contain an activity that can phosphorylate MAP kinase, suggesting that Mos directly or indirectly activates a MAPKK. Furthermore, activated MBP-Mos fusion protein is able to phosphorylate and activate a purified, phosphatase-treated, rabbit muscle MAPKK in vitro. Thus, in oocytes, Mos is an upstream activator of MAP kinase which may function through direct phosphorylation of MAPKK.

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Feilmeier, Bradley J., Ginger Iseminger, Diane Schroeder, Hannali Webber, and Gregory J. Phillips. "Green Fluorescent Protein Functions as a Reporter for Protein Localization in Escherichia coli." Journal of Bacteriology 182, no. 14 (July 15, 2000): 4068–76. http://dx.doi.org/10.1128/jb.182.14.4068-4076.2000.

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ABSTRACT The use of green fluorescent protein (GFP) as a reporter for protein localization in Escherichia coli was explored by creating gene fusions between malE (encoding maltose-binding protein [MBP]) and a variant of gfpoptimized for fluorescence in bacteria (GFPuv). These constructs encode hybrid proteins composed of GFP fused to the carboxy-terminal end of MBP. Fluorescence was not detected when the hybrid protein was synthesized with the MBP signal sequence. In contrast, when the MBP signal sequence was deleted, fluorescence was observed. Cell fractionation studies showed that the fluorescent MBP-GFP hybrid protein was localized in the cytoplasm, whereas the nonfluorescent version was localized to the periplasmic space. Smaller MBP-GFP hybrid proteins, however, exhibited abnormal fractionation. Expression of the gene fusions in different sec mutants, as well as signal sequence processing assays, confirmed that the periplasmically localized hybrid proteins were exported by thesec-dependent pathway. The distinction between fluorescent and nonfluorescent colonies was exploited as a scorable phenotype to isolate malE signal sequence mutations. While expression of hybrid proteins comprised of full-length MBP did not result in overproduction lethality characteristic of some exported β-galactosidase hybrid proteins, synthesis of shorter, exported hybrid proteins was toxic to the cells. Purification of MBP-GFP hybrid protein from the different cellular compartments indicated that GFP is improperly folded when localized outside of the cytoplasm. These results suggest that GFP could serve as a useful reporter for genetic analysis of bacterial protein export and of protein folding.

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Haurum, J. S., S. Thiel, H. P. Haagsman, S. B. Laursen, B. Larsen, and J. C. Jensenius. "Studies on the carbohydrate-binding characteristics of human pulmonary surfactant-associated protein A and comparison with two other collectins: mannan-binding protein and conglutinin." Biochemical Journal 293, no. 3 (August 1, 1993): 873–78. http://dx.doi.org/10.1042/bj2930873.

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The surfactant-associated protein A (SP-A) belongs to the collectin family, a group of C-type lectins encompassing also surfactant-associated protein D, mannan-binding protein (MBP) and conglutinin. These proteins all have carbohydrate-recognition domains joined to collagen stalks. It seems likely that SP-A, like MBP and conglutinin, may mediate anti-microbial activity through binding to carbohydrates on the microorganisms and collectin receptors on phagocytic cells. We have studied the influence of carbohydrates on the binding of SP-A, MBP and conglutinin to mannan in an enzyme-linked lectin-binding assay. All sugars were of D-configuration, except fucose of which both L- and D-configurations were tested. The order of inhibiting potency on the binding of SP-A was: N-acetylmannosamine > L-fucose, maltose > glucose > mannose. The following sugars were non-inhibitory: galactose, D-fucose, glucosamine, mannosamine, galactosamine, N-acetylglucosamine, and N-acetylgalactosamine. The best inhibitor of MBP was N-acetylglucosamine. Otherwise MBP showed a selectivity similar to that of SP-A. Conglutinin binding was inhibited by all the sugars examined except N-acetylgalactosamine. For conglutinin, as for MBP, the best inhibitor was N-acetylglucosamine. Normal human SP-A, alveolar-proteinosis SP-A purified by ion-exchange chromatography, and alveolar-proteinosis SP-A purified by n-butanol extraction showed no difference in sugar selectivity. The influence of pH and of the calcium concentration was also examined. Organic solvent-extracted SP-A from patients suffering from alveolar proteinosis and normal SP-A showed different sensitivity profiles.

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Rico, Ana I., Gustavo Del Real, Manuel Soto, Luis Quijada, Carlos Martinez-A., Carlos Alonso, and Jose M. Requena. "Characterization of the Immunostimulatory Properties of Leishmania infantum HSP70 by Fusion to theEscherichia coli Maltose-Binding Protein in Normal andnu/nu BALB/c Mice." Infection and Immunity 66, no. 1 (January 1, 1998): 347–52. http://dx.doi.org/10.1128/iai.66.1.347-352.1998.

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ABSTRACT Leishmania infantum HSP70 has been described as an immunodominant antigen in both humans and dogs suffering from visceral leishmaniasis. In this study, we used L. infantum HSP70 fused to Escherichia coli maltose-binding protein (MBP), as the reporter protein, to analyze the influence of HSP70 on the immunogenicity of MBP in BALB/c mice. Plasmids were constructed to produce the three recombinant proteins used in this study, namely, MBP,L. infantum HSP70, and MBP-HSP70, which consists of MBP fused to the L. infantum HSP70 amino terminus. Immunization of BALB/c mice with the MBP-HSP70 fusion protein elicited humoral and cellular responses against MBP that were higher by an order of magnitude than those elicited by immunization with MBP alone or with a mixture of MBP and HSP70. Covalent linkage of MBP to HSP70 was essential for eliciting a strong anti-MBP immune response. Cytokine secretion and immunoglobulin G isotype analyses indicated that immunization with the MBP-HSP70 fusion protein preferentially induces a Th1 immune response. Immunization of athymic nu/nu mice with the MBP-HSP70 fusion protein unexpectedly gave rise to an anti-MBP humoral response showing features of a T-cell-dependent response. Thus, we present evidence that L. infantum HSP70 demonstrates an adjuvant effect in the immune response against a covalently linked reporter protein.

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BUCHE, André, Carmen MÉNDEZ, and José A. SALAS. "Interaction between ATP, oleandomycin and the OleB ATP-binding cassette transporter of Streptomyces antibioticus involved in oleandomycin secretion." Biochemical Journal 321, no. 1 (January 1, 1997): 139–44. http://dx.doi.org/10.1042/bj3210139.

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The OleB protein of Streptomyces antibioticus, oleandomycin (OM) producer, constitutes an ATP-binding cassette transporter containing two nucleotide-binding domains and is involved in OM resistance and its secretion in this producer strain. We have characterized some properties of the first nucleotide-binding domain of OleB using an overexpressed fusion protein (MBP–OleB´) between a maltose-binding protein (MBP) and the first half of OleB (OleB´). Extrinsic fluorescence of the base-modified fluorescent nucleotide analogue 1,N6-ethenoadenosine 5´-triphosphate (εATP) and 2´(3´)-o-(2,4,6-trinitrophenyl)adenosine-5´-triphosphate was determined in the presence of MBP and the fusion protein MBP–OleB´, and it was found that εATP binds to MBP–OleB´ with a stoichiometry of 0.9. Measurements of the intrinsic fluorescence of the MBP–OleB´ fusion protein indicated that ATP induces a decrease in the accessibility of the MBP–OleB´ tryptophans to acrylamide, an indication of a folding effect. This conclusion was confirmed by the fact that ATP also induces considerable stabilization against guanidine chloride denaturation of MBP–OleB´. Two effects were found to be associated with the presence of Mg2+ ions: (1) an increase in the quenching of MBP–OleB´ intrinsic fluorescence by ATP; and (2) an increase in the accessibility of MBP–OleB´ tryptophans to acrylamide. Significant changes in the intrinsic fluorescence of the fusion protein were also observed in the presence of OM, demostrating the existence of interaction between the transporter and the antibiotic in the absence of any hydrophobic membrane component.

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Tinsley, Eowyn, Asma Naqvi, Agathe Bourgogne, Theresa M. Koehler, and Saleem A. Khan. "Isolation of a Minireplicon of the Virulence Plasmid pXO2 of Bacillus anthracis and Characterization of the Plasmid-Encoded RepS Replication Protein." Journal of Bacteriology 186, no. 9 (May 1, 2004): 2717–23. http://dx.doi.org/10.1128/jb.186.9.2717-2723.2004.

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ABSTRACT A minireplicon of plasmid pXO2 of Bacillus anthracis was isolated by molecular cloning in Escherichia coli and shown to replicate in B. anthracis, Bacillus cereus, and Bacillus subtilis. The pXO2 replicon included (i) an open reading frame encoding the putative RepS replication initiation protein and (ii) the putative origin of replication. The RepS protein was expressed as a fusion with the maltose binding protein (MBP) at its amino-terminal end and purified by affinity chromatography. Electrophoretic mobility shift assays showed that the purified MBP-RepS protein bound specifically to a 60-bp region corresponding to the putative origin of replication of pXO2 located immediately downstream of the RepS open reading frame. Competition DNA binding experiments showed that the 5′ and central regions of the putative origin were important for RepS binding. MBP-RepS also bound nonspecifically to single-stranded DNA with a lower affinity.

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Nickolaus, Chen, Carolyn Vargas, Jörg Reichenwallner, Mohammed Chakour, Benjamin Selmke, Rusha Chakraborty, Raghavan Varadarajan, Sandro Keller, and Wolfgang E. Trommer. "The Molten Globule State of Maltose-Binding Protein: Structural and Thermodynamic Characterization by EPR Spectroscopy and Isothermal Titration Calorimetry." Applied Magnetic Resonance 51, no. 9-10 (September 22, 2020): 877–86. http://dx.doi.org/10.1007/s00723-020-01232-y.

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Abstract Employing site-directed spin labeling (SDSL), the structure of maltose-binding protein (MBP) had previously been studied in the native state by electron paramagnetic resonance (EPR) spectroscopy. Several spin-labeled double cysteine mutants were distributed all over the structure of this cysteine-free protein and revealed distance information between the nitroxide residues from double electron–electron resonance (DEER). The results were in good agreement with the known X-ray structure. We have now extended these studies to the molten globule (MG) state, a folding intermediate, which can be stabilized around pH 3 and that is characterized by secondary but hardly any tertiary structure. Instead of clearly defined distance features as found in the native state, several additional characteristics indicate that the MG structure of MBP contains different polypeptide chain and domain orientations. MBP is also known to bind its substrate maltose even in MG state although with lower affinity. Additionally, we have now created new mutants allowing for spin labeling at or near the active site. Our data confirm an already preformed ligand site structure in the MG explaining its substrate binding capability and thus most probably serving as a nucleation center for the final native structure.

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WANG, Changsen, Ariel F. CASTRO, Denise M. WILKES, and Guillermo A. ALTENBERG. "Expression and purification of the first nucleotide-binding domain and linker region of human multidrug resistance gene product: comparison of fusions to glutathione S-transferase, thioredoxin and maltose-binding protein." Biochemical Journal 338, no. 1 (February 8, 1999): 77–81. http://dx.doi.org/10.1042/bj3380077.

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Many membrane proteins that belong to the ATP-binding cassette (ABC) superfamily are clinically important, including the cystic fibrosis transmembrane conductance regulator, the sulphonylurea receptor and P-glycoprotein (multidrug resistance gene product; MDR1). These proteins contain two multispanning transmembrane domains, each followed by one nucleotide-binding domain (NBD) and a linker region distal to the first NBD. ATP hydrolysis by the NBDs is critical for ABC protein function; the linker region seems to have a regulatory role. Previous attempts to express soluble NBDs and/or linker regions without detergent solubilization, or to purify NBDs at high yields as soluble fusion proteins, have been unsuccessful. Here we present a system for the expression in Escherichia coli of the first NBD of MDR1 followed by its linker region (NBD1MLD). A comparison of the expressions of NBD1MLD fused to glutathione S-transferase, thioredoxin and maltose-binding protein (MBP) shows that a high level of expression in the soluble fraction (approx. 8% of total E. coli protein) can be achieved only for MBP–NBD1MLD. The addition of a proteolytic thrombin site just proximal to the N-terminal end of NBD1MLD allows the cleavage of NBD1MLD from MBP, which can be easily purified with retention of its ATPase activity. In summary, success was obtained only when using an MBP fusion protein vector containing a thrombin proteolytic site between MBP and NBD1MLD. The approach described here could be generally applicable to solving the problems of expression and purification of NBDs/linker regions of ABC proteins.

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Zhao, Xinyu, Guoshun Li, and Shufang Liang. "Several Affinity Tags Commonly Used in Chromatographic Purification." Journal of Analytical Methods in Chemistry 2013 (2013): 1–8. http://dx.doi.org/10.1155/2013/581093.

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Affinity tags have become powerful tools from basic biological research to structural and functional proteomics. They were widely used to facilitate the purification and detection of proteins of interest, as well as the separation of protein complexes. Here, we mainly discuss the benefits and drawbacks of several affinity or epitope tags frequently used, including hexahistidine tag, FLAG tag, Strep II tag, streptavidin-binding peptide (SBP) tag, calmodulin-binding peptide (CBP), glutathione S-transferase (GST), maltose-binding protein (MBP), S-tag, HA tag, and c-Myc tag. In some cases, a large-size affinity tag, such as GST or MBP, can significantly impact on the structure and biological activity of the fusion partner protein. So it is usually necessary to excise the tag by protease. The most commonly used endopeptidases are enterokinase, factor Xa, thrombin, tobacco etch virus, and human rhinovirus 3C protease. The proteolysis features of these proteases are described in order to provide a general guidance on the proteolytic removal of the affinity tags.

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Imai, Noriko, Noriyuki Matsuda, Keiji Tanaka, Akihiko Nakano, Shogo Matsumoto, and WonKyung Kang. "Ubiquitin Ligase Activities of Bombyx mori Nucleopolyhedrovirus RING Finger Proteins." Journal of Virology 77, no. 2 (January 15, 2003): 923–30. http://dx.doi.org/10.1128/jvi.77.2.923-930.2003.

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ABSTRACT The genome of Bombyx mori nucleopolyhedrovirus (BmNPV) is predicted to contain six RING finger proteins: IAP1, ORF35, IAP2, CG30, IE2, and PE38. Several other members of the RING finger family have recently been shown to have the ubiquitin-ligase (E3) activity. We thus examined whether BmNPV RING finger proteins have the E3 activity. In vitro ubiquitination assay with the rabbit reticulocyte lysates and BmNPV RING finger proteins fused with maltose-binding protein (MBP) showed that four of them (IAP2, IE2, PE38, and CG30) were polyubiquitinated in the presence of zinc ion. Furthermore, MBP-IAP2, MBP-IE2, and MBP-PE38 were able to reconstitute ubiquitination activity in cooperation with the Ubc4/5 subfamily of ubiquitin-conjugating enzymes. Mutational analysis also showed that ubiquitination activity of MBP-IAP2, MBP-IE2, and MBP-PE38 were dependent on their RING finger motif. Therefore, these results suggest that IAP2, IE2, and PE38 may function as E3 enzymes during BmNPV infection.

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Vu, Thu Trang Thi, Boram Jeong, Martin Krupa, Uijung Kwon, Jung-A. Song, Bich Hang Do, Minh Tan Nguyen, et al. "Soluble Prokaryotic Expression and Purification of Human Interferon Alpha-2b Using a Maltose-Binding Protein Tag." Journal of Molecular Microbiology and Biotechnology 26, no. 6 (2016): 359–68. http://dx.doi.org/10.1159/000446962.

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Human interferon alpha-2b (IFNα-2b) has therapeutic applications as an antiviral and antiproliferative drug and has been used for a wide range of indications. Efficient production of IFNα-2b in Escherichia coli has been difficult because the protein tends to form inclusion bodies. This obstacle has garnered interest in efficiently expressing IFNα-2b and overcoming its poor solubility. In this study, seven N-terminal fusion partners - hexahistidine (His6), thioredoxin, glutathione S-transferase (GST), maltose-binding protein (MBP), N-utilization substance protein A, protein disulfide bond isomerase (PDI), and b'a' domain of PDI - were tested for soluble overexpression of codon-optimized IFNα-2b in E. coli. Low temperature increased the expression level of all of the tagged proteins except for the GST fusion. All the tags, except for His6 and GST, improved solubility. We purified IFNα-2b from the MBP-tagged fusion using immobilized metal affinity chromatography and anion exchange chromatography, and obtained a final yield of 7.2 mg from an initial 500-ml culture. The endotoxin level was 0.46 EU/µg. Biological activity was demonstrated using a luciferase assay, which showed a dose-dependent response with a calculated EC50 of 10.3 ± 5.9 p<smlcap>M</smlcap>. Our results demonstrate that using an MBP-tagged fusion is an efficient way to produce pure IFNα-2b.

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Zhou, Lilin, Jingjun Wu, Huijuan Zhang, Yan Kang, Jun Guo, Chong Zhang, Jinying Yuan, and Xinhui Xing. "Magnetic nanoparticles for the affinity adsorption of maltose binding protein (MBP) fusion enzymes." Journal of Materials Chemistry 22, no. 14 (2012): 6813. http://dx.doi.org/10.1039/c2jm16778f.

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Aggarwal, Vasudha, Rajendra Kulothungan S., Saranya Rajaram, Balamurali M.M., Raghavan Varadarajan, and Sri Rama Koti Ainavarapu. "Single-Molecule Studies of the Parallel Unfolding Pathways of Maltose Binding Protein (MBP)." Biophysical Journal 100, no. 3 (February 2011): 481a. http://dx.doi.org/10.1016/j.bpj.2010.12.2821.

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34

Fang, Fugui, Ya Liu, Yong Pu, Lin Wang, Suolu Wang, and Xiaorong Zhang. "Immunogenicity of Recombinant Maltose-binding Protein (MBP)–Gonadotropin Releasing Hormone I (GnRH-I)." Systems Biology in Reproductive Medicine 56, no. 6 (September 17, 2010): 478–86. http://dx.doi.org/10.3109/19396368.2010.481005.

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35

KISHORE, Uday, Jiu-Yao WANG, Hans-Jürgen HOPPE, and Kenneth B. M. REID. "The α-helical neck region of human lung surfactant protein D is essential for the binding of the carbohydrate recognition domains to lipopolysaccharides and phospholipids." Biochemical Journal 318, no. 2 (September 1, 1996): 505–11. http://dx.doi.org/10.1042/bj3180505.

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We have expressed the carbohydrate recognition domains (CRDs) of human lung surfactant protein D (SP-D) in Escherichia coli as a trimeric structure held together by the α-helical neck region of the molecule. The DNA sequence coding for the neck-region peptide and the CRD of SP-D was subcloned and expressed as a fusion protein containing the E. coli maltose binding protein (MBP). After removal of the MBP, the recombinant structure, containing three CRDs of SP-D, was found to be comparable to native SP-D in terms of carbohydrate binding specificity, the binding to lipopolysaccharides (LPSs) of Gram-negative bacteria, and interaction with phospholipids. The CRD of SP-D, without the neck region peptide, was also expressed and shown to behave as a monomer that showed a very weak affinity for maltose-agarose, LPSs and phospholipids. The α-helical neck region on its own showed affinity for phospholipids and thus might contribute to the binding of SP-D to these structures. However, the possibility that hydrophobic patches, which are exposed only in the isolated neck region and not in the intact SP-D, plays a role in neck region–phospholipid interaction, cannot be excluded. The results confirm the importance of the neck region as a trimerizing agent in bringing together three CRDs and suggest that multivalency is important in the strong binding of SP-D to carbohydrate targets.

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Collier, D. N., S. M. Strobel, and P. J. Bassford. "SecB-independent export of Escherichia coli ribose-binding protein (RBP): some comparisons with export of maltose-binding protein (MBP) and studies with RBP-MBP hybrid proteins." Journal of Bacteriology 172, no. 12 (1990): 6875–84. http://dx.doi.org/10.1128/jb.172.12.6875-6884.1990.

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Ishii, Yoshiyuki, Kaoru Murakami, Hiroaki I.-Ogawa, Akihiko Kondo, and Yasuhiko Kato. "Production of MBP(maltose binding protein)-GroES fusion protein and utilization to stimulate GroEL-mediated protein refolding." Journal of Fermentation and Bioengineering 85, no. 1 (January 1998): 69–73. http://dx.doi.org/10.1016/s0922-338x(97)80356-x.

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Watterson, Daniel, Bostjan Kobe, and Paul R. Young. "Residues in domain III of the dengue virus envelope glycoprotein involved in cell-surface glycosaminoglycan binding." Journal of General Virology 93, no. 1 (January 1, 2012): 72–82. http://dx.doi.org/10.1099/vir.0.037317-0.

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The dengue virus (DENV) envelope (E) protein mediates virus entry into cells via interaction with a range of cell-surface receptor molecules. Cell-surface glycosaminoglycans (GAGs) have been shown to play an early role in this interaction, and charged oligosaccharides such as heparin bind to the E protein. We have examined this interaction using site-directed mutagenesis of a recombinant form of the putative receptor-binding domain III of the DENV-2E protein expressed as an MBP (maltose-binding protein)-fusion protein. Using an ELISA-based GAG-binding assay, cell-based binding analysis and antiviral-activity assays, we have identified two critical residues, K291 and K295, that are involved in GAG interactions. These studies have also demonstrated differential binding between mosquito and human cells.

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Tong, Junsen, Huiseon Yang, and Young Jun Im. "Crystallization and preliminary X-ray crystallographic analysis of the C-terminal domain of guanylate kinase-associated protein fromRattus norvegicus." Acta Crystallographica Section F Structural Biology Communications 70, no. 7 (June 19, 2014): 949–54. http://dx.doi.org/10.1107/s2053230x1401187x.

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Guanylate kinase-associated protein (GKAP) is a scaffolding protein that plays a role in protein–protein interactions at the synaptic junction such as linking the NMDA receptor–PSD-95 complex to the Shank–Homer complex. In this study, the C-terminal helical domain of GKAP fromRattus norvegicuswas purified and crystallized by the vapour-diffusion method. To improve the diffraction quality of the GKAP crystals, a flexible loop in GKAP was truncated and an MBP (maltose-binding protein)-GKAP fusion was constructed in which the last C-terminal helix of MBP is fused to the N-terminus of the GKAP domain. The MBP-GKAP crystals diffracted to 2.0 Å resolution using synchrotron radiation. The crystal was orthorhombic, belonging to space groupP21212, with unit-cell parametersa= 99.1,b= 158.7,c= 65.5 Å. The Matthews coefficient was determined to be 2.44 Å3 Da−1(solvent content 49.5%) with two molecules in the asymmetric unit. Initial attempts to solve the structure by molecular replacement using the MBP structure were successful.

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Zhang, Dan-Ping, Xiao-Ran Jing, An-Wen Fan, Huan Liu, Yao Nie, and Yan Xu. "Active Expression of Membrane-Bound L-Amino Acid Deaminase from Proteus mirabilis in Recombinant Escherichia coli by Fusion with Maltose-Binding Protein for Enhanced Catalytic Performance." Catalysts 10, no. 2 (February 10, 2020): 215. http://dx.doi.org/10.3390/catal10020215.

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L-amino acid deaminases (LAADs) are membrane flavoenzymes that catalyze the deamination of neutral and aromatic L-amino acids to α-keto acids and ammonia. LAADs can be used to develop many important biotechnological applications. However, the transmembrane α-helix of LAADs restricts its soluble active expression and purification from a heterologous host, such as Escherichia coli. Herein, through fusion with the maltose-binding protein (MBP) tag, the recombinant E. coli BL21 (DE3)/pET-21b-MBP-PmLAAD was constructed and the LAAD from Proteus mirabilis (PmLAAD) was actively expressed as a soluble protein. After purification, the purified MBP-PmLAAD was obtained. Then, the catalytic activity of the MBP-PmLAAD fusion protein was determined and compared with the non-fused PmLAAD. After fusion with the MBP-tag, the catalytic efficiency of the MBP-PmLAAD cell lysate was much higher than that of the membrane-bound PmLAAD whole cells. The soluble MBP-PmLAAD cell lysate catalyzed the conversion of 100 mM L-phenylalanine (L-Phe) to phenylpyruvic acid (PPA) with a 100% yield in 6 h. Therefore, the fusion of the MBP-tag not only improved the soluble expression of the PmLAAD membrane-bound protein, but also increased its catalytic performance.

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Webber, Tawnya, Sarsati Gurung, Justin Saul, Trenton Baker, Michelle Spatara, Matthew Freyer, Anne Skaja Robinson, and Matthew J. Gage. "The C-terminus of the P22 tailspike protein acts as an independent oligomerization domain for monomeric proteins." Biochemical Journal 419, no. 3 (April 14, 2009): 595–602. http://dx.doi.org/10.1042/bj20081449.

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TSP (P22 tailspike protein) is a well-established model system for studying the folding and assembly of oligomeric proteins, and previous studies have documented both in vivo and in vitro folding intermediates using this protein. Especially important is the C-terminus of TSP, which plays a critical role in the assembly and maturation of the protrimer intermediate to its final trimeric form. In the present study, we show that by grafting the C-terminus of TSP on to the monomeric MBP (maltose-binding protein), the resulting chimaera (MBP-537) is a trimeric protein. Moreover, Western blot studies (using an anti-TSP antibody) indicate that the TSP C-terminus in the MBP-537 chimaera has the same conformation as the native TSP. The oligomerization of the MBP-537 chimaera appears to involve hydrophobic interactions and a refolding sequence, both of which are analogous to the native TSP. These results underscore the importance of the TSP C-terminus in the assembly of the mature trimer and demonstrate its potential utility as a model to study the folding and assembly of the TSP C-terminus in isolation.

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Matsuda, N., T. Suzuki, K. Tanaka, and A. Nakano. "Rma1, a novel type of RING finger protein conserved from Arabidopsis to human, is a membrane-bound ubiquitin ligase." Journal of Cell Science 114, no. 10 (May 15, 2001): 1949–57. http://dx.doi.org/10.1242/jcs.114.10.1949.

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Rma1 is a protein with a RING finger motif and a C-terminal membrane-anchoring domain and is well conserved among higher eukaryotes. We show that fusion proteins between maltose binding protein (MBP) and human or Arabidopsis Rma1 are polyubiquitinated, when incubated with the rabbit reticulocyte or the wheat germ lysate, respectively. The polyubiquitination of MBP-Rma1 has been reconstituted by incubation with purified ubiquitin, the ubiquitin-activating enzyme E1, and one of the two ubiquitin-conjugating E2 enzymes (Ubc4 or UbcH5a). Other E2 enzymes tested, E2-20k, E2-25k, Ubc3 and Ubc8, are not able to confer this modification. Mutational analysis shows that the RING finger motif of Rma1 is necessary for the auto-ubiquitination of MBP-Rma1. Thus, Rma1 represents a novel, membrane-bound type of ubiquitin ligase E3, which probably functions with the Ubc4/5 subfamily of E2. The MBP moiety but not Rma1 itself is ubiquitinated in the auto-ubiquitination reaction of MBP-Rma1. Free MBP in solution is not a substrate of Rma1. These observations indicate that bringing the substrate into its physical vicinity is very important for the action of ubiquitin ligase.

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Park, Jung-Hyun, Shin-Young Na, Dong Gun Lee, Byoung-Don Han, and Kil Lyong Kim. "Single-step purification of proteins of interest from proteolytically cleaved recombinant maltose-binding protein (MBP) fusion proteins by selective immunoprecipitation of MBP." Biotechnology and Bioprocess Engineering 3, no. 2 (December 1998): 82–86. http://dx.doi.org/10.1007/bf02932507.

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Park, Sangsu, Minh Quan Nguyen, Huynh Kim Khanh Ta, Minh Tan Nguyen, Gunsup Lee, Chong Jai Kim, Yeon Jin Jang, and Han Choe. "Soluble Cytoplasmic Expression and Purification of Immunotoxin HER2(scFv)-PE24B as a Maltose Binding Protein Fusion." International Journal of Molecular Sciences 22, no. 12 (June 17, 2021): 6483. http://dx.doi.org/10.3390/ijms22126483.

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Human epidermal growth factor receptor 2 (HER-2) is overexpressed in many malignant tumors. The anti-HER2 antibody trastuzumab has been approved for treating HER2-positive early and metastatic breast cancers. Pseudomonas exotoxin A (PE), a bacterial toxin of Pseudomonas aeruginosa, consists of an A-domain with enzymatic activity and a B-domain with cell binding activity. Recombinant immunotoxins comprising the HER2(scFv) single-chain Fv from trastuzumab and the PE24B catalytic fragment of PE display promising cytotoxic effects, but immunotoxins are typically insoluble when expressed in the cytoplasm of Escherichia coli, and thus they require solubilization and refolding. Herein, a recombinant immunotoxin gene was fused with maltose binding protein (MBP) and overexpressed in a soluble form in E. coli. Removal of the MBP yielded stable HER2(scFv)-PE24B at 91% purity; 0.25 mg of pure HER2(scFv)-PE24B was obtained from a 500 mL flask culture. Purified HER2(scFv)-PE24B was tested against four breast cancer cell lines differing in their surface HER2 level. The immunotoxin showed stronger cytotoxicity than HER2(scFv) or PE24B alone. The IC50 values for HER2(scFv)-PE24B were 28.1 ± 2.5 pM (n = 9) and 19 ± 1.4 pM (n = 9) for high HER2-positive cell lines SKBR3 and BT-474, respectively, but its cytotoxicity was lower against MDA-MB-231 and MCF7. Thus, fusion with MBP can facilitate the soluble expression and purification of scFv immunotoxins.

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Nickel, Osmar, Maria L. P. N. Targon, Thor V. M. Fajardo, Marcos A. Machado, and Ana P. Trivilin. "Polyclonal antibodies to the coat protein of Apple stem grooving virus expressed in Escherichia coli: production and use in immunodiagnosis." Fitopatologia Brasileira 29, no. 5 (October 2004): 558–62. http://dx.doi.org/10.1590/s0100-41582004000500017.

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The coat protein gene of Apple stem grooving virus (ASGV) was amplified by RT-PCR, cloned, sequenced and subcloned in the expression vector pMal-c2. This plasmid was used to transform Escherichia coli BL21c+ competent cells. The ASGV coat protein (cp) was expressed as a fusion protein containing a fragment of E. coli maltose binding protein (MBP). Bacterial cells were disrupted by sonication and the ASGVcp/MBP fusion protein was purified by amylose resin affinity chromatography. Polyclonal antibodies from rabbits immunized with the fusion protein gave specific reactions to ASGV from infected apple (Malus domestica) cv. Fuji Irradiada and Chenopodium quinoa at dilutions of up to 1:1,000 and 1:2,000, respectively, in plate trapped ELISA. The ASGVcp/MBP fusion protein reacted to a commercial antiserum against ASGV in immunoblotting assay. The IgG against ASGVcp/MBP performed favorably in specificity and sensitivity to the virus. This method represents an additional tool for the efficient ASGV-indexing of apple propagative and mother stock materials, and for use in support of biological and molecular techniques.

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Taylor, Abigail, Mark Warner, Christopher Mendoza, Calvin Memmott, Tom LeCheminant, Sara Bailey, Colter Christensen, Julie Keller, Arminda Suli, and Dario Mizrachi. "Chimeric Claudins: A New Tool to Study Tight Junction Structure and Function." International Journal of Molecular Sciences 22, no. 9 (May 6, 2021): 4947. http://dx.doi.org/10.3390/ijms22094947.

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The tight junction (TJ) is a structure composed of multiple proteins, both cytosolic and membranal, responsible for cell–cell adhesion in polarized endothelium and epithelium. The TJ is intimately connected to the cytoskeleton and plays a role in development and homeostasis. Among the TJ’s membrane proteins, claudins (CLDNs) are key to establishing blood–tissue barriers that protect organismal physiology. Recently, several crystal structures have been reported for detergent extracted recombinant CLDNs. These structural advances lack direct evidence to support quaternary structure of CLDNs. In this article, we have employed protein-engineering principles to create detergent-independent chimeric CLDNs, a combination of a 4-helix bundle soluble monomeric protein (PDB ID: 2jua) and the apical—50% of human CLDN1, the extracellular domain that is responsible for cell–cell adhesion. Maltose-binding protein-fused chimeric CLDNs (MBP-CCs) used in this study are soluble proteins that retain structural and functional aspects of native CLDNs. Here, we report the biophysical characterization of the structure and function of MBP-CCs. MBP-fused epithelial cadherin (MBP-eCAD) is used as a control and point of comparison of a well-characterized cell-adhesion molecule. Our synthetic strategy may benefit other families of 4-α-helix membrane proteins, including tetraspanins, connexins, pannexins, innexins, and more.

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Smith, Richard H., and Robert M. Kotin. "The Rep52 Gene Product of Adeno-Associated Virus Is a DNA Helicase with 3′-to-5′ Polarity." Journal of Virology 72, no. 6 (June 1, 1998): 4874–81. http://dx.doi.org/10.1128/jvi.72.6.4874-4881.1998.

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ABSTRACT The rep gene of adeno-associated virus type 2 encodes four overlapping proteins from two separate promoters, termed P5 and P19. The P5-promoted Rep proteins, Rep78 and Rep68, are essential for viral DNA replication, and a wealth of data concerning the biochemical activities of these proteins has been reported. In contrast, data concerning the biochemical functions of the P19-promoted Rep proteins, Rep52 and Rep40, are lacking. Here, we describe enzymatic activities associated with a bacterially expressed maltose-binding protein (MBP)-Rep52 fusion protein. Purified MBP-Rep52 possesses 3′-to-5′ DNA helicase activity that is strictly dependent upon the presence of nucleoside triphosphate and divalent cation cofactors. In addition, MBP-Rep52 demonstrates a constitutive ATPase activity that is active in the absence of DNA effector molecules. An MBP-Rep52 chimera bearing a lysine-to-histidine substitution at position 116 (K116H) within a consensus helicase- and ATPase-associated motif (motif I or Walker A site) was deficient for both DNA helicase and ATPase activities. In contrast to a Rep78 A-site mutant protein bearing a corresponding amino acid substitution at position 340 (K340H), the MBP-Rep52 A-site mutant protein failed to exhibit a trans-dominant negative effect when it was mixed with wild-type MBP-Rep52 or MBP-Rep78 in vitro. This lack of trans dominance, coupled with the results of coimmunoprecipitation and gel filtration chromatography experiments reported here, suggests that the ability of Rep52 to engage in multimeric interactions may differ from that of Rep78 or -68.

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Zhou, Hongyue, Zenan Zhang, Guomu Liu, Mengyu Jiang, Jingjing Wang, Yu Liu, and Guixiang Tai. "The Effect of Different Immunization Cycles of a Recombinant Mucin1-Maltose-Binding Protein Vaccine on T Cell Responses to B16-MUC1 Melanoma in Mice." International Journal of Molecular Sciences 21, no. 16 (August 13, 2020): 5810. http://dx.doi.org/10.3390/ijms21165810.

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We explored the effect of a recombinant mucin1-maltose-binding protein vaccine, including immunization cycles of recombinant mucin1-maltose-binding protein (MUC1-MBP) and CpG 2006 on T cell responses to human MUC1-overexpressing mouse melanoma B16 cells (B16-MUC1) melanoma in mice. We found that the vaccine had a significant antitumor effect, with the most obvious tumor-suppressive effect being observed in mice immunized five times. After more than five immunizations, the tumor inhibition rate decreased from 81.67% (five immunizations) to 43.67% (eight immunizations). To study the possible mechanism, Mucin-1(MUC1)-specific antibodies, IFN-γ secretion by lymphocytes, and cytotoxic T lymphocyte (CTL) cytotoxicity were measured by enzyme-linked immunosorbent assay (ELISA) and a real-time cell analyzer (RTCA). T cell subsets and immunosuppressive cells in the mouse spleen and tumor microenvironment were analyzed by FACS. These results showed that five immunizations activated MUC1-specific Th1 and CTL and reduced the ratio of myeloid-derived suppressor cells (MDSCs) and Th17 in mice more significantly than eight immunizations, indicating that excessive frequency of the immune cycle leads to the increased numbers of immunosuppressive cells and decreased numbers of immunostimulatory cells, thereby inhibiting antitumor immune activity. This data provide an experimental foundation for the clinical application of a recombinant MUC1-MBP vaccine.

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Smith, Richard H., and Robert M. Kotin. "An Adeno-Associated Virus (AAV) Initiator Protein, Rep78, Catalyzes the Cleavage and Ligation of Single-Stranded AAV ori DNA." Journal of Virology 74, no. 7 (April 1, 2000): 3122–29. http://dx.doi.org/10.1128/jvi.74.7.3122-3129.2000.

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ABSTRACT The Rep78 protein of adeno-associated virus (AAV) contains amino acid sequence motifs common to rolling-circle replication (RCR) initiator proteins. In this report, we describe RCR initiator-like activities of Rep78. We demonstrate that a maltose-binding protein (MBP)–Rep78 fusion protein can catalyze the cleavage and ligation of single-stranded DNA substrates derived from the AAV origin of replication. Rep-mediated single-stranded DNA cleavage was strictly dependent on the presence of certain divalent cations (e.g., Mn2+ or Mg2+) but did not require the presence of a nucleoside triphosphate cofactor. Electrophoretic mobility shift assays demonstrated that binding of single-stranded DNA by MBP-Rep78 was influenced by the length of the substrate as well as the presence of potential single-stranded cis-acting sequence elements. Site-directed mutagenesis was used to examine the role of specific tyrosine residues within a conserved RCR motif (motif 3) of Rep78. Replacement of Tyr-156 with phenylalanine abolished the ability of MBP-Rep78 to mediate the cleavage and ligation of single-stranded DNA substrates but not the ability to stably bind single-stranded DNA. The cleaving-joining activity of Rep78 is consistent with the mechanism of replicative intermediate dimer resolution proposed for the autonomous parvoviruses and may have implications for targeted integration of recombinant AAV vectors.

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Gannon, P. M., P. Li, and C. A. Kumamoto. "The mature portion of Escherichia coli maltose-binding protein (MBP) determines the dependence of MBP on SecB for export." Journal of Bacteriology 171, no. 2 (1989): 813–18. http://dx.doi.org/10.1128/jb.171.2.813-818.1989.

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Journal articles on the topic 'Maltose binding protein (MBP)'

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