Escherichia coli maltose‐binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused

Although it is usually possible to achieve a favorable yield of a recombinant protein in Escherichia coli, obtaining the protein in a soluble, biologically active form continues to be a major challenge. Sometimes this problem can be overcome by fusing an aggregation‐prone polypeptide to a highly soluble partner. To study this phenomenon in greater detail, we compared the ability of three soluble fusion partners—maltose‐binding protein (MBP), glutathione S‐transferase (GST), and thioredoxin (TRX)—to inhibit the aggregation of six diverse proteins that normally accumulate in an insoluble form. Remarkably, we found that MBP is a far more effective solubilizing agent than the other two fusion partners. Moreover, we demonstrated that in some cases fusion to MBP can promote the proper folding of the attached protein into its biologically active conformation. Thus, MBP seems to be capable of functioning as a general molecular chaperone in the context of a fusion protein. A model is proposed to explain how MBP promotes the solubility and influences the folding of its fusion partners.

[1]  A. Reddy,et al.  High-level expression of the Endo-beta-N-acetylglucosaminidase F2 gene in E.coli: one step purification to homogeneity. , 1998, Glycobiology.

[2]  T. Aoki,et al.  Purification of recombinant human pepsinogens and their application as immunoassay standards , 1998, Biochemistry and molecular biology international.

[3]  J. Chirgwin,et al.  Order of fusions between bacterial and mammalian proteins can determine solubility in Escherichia coli. , 1998, Biochemical and biophysical research communications.

[4]  Yuan Zhang,et al.  Expression of eukaryotic proteins in soluble form in Escherichia coli. , 1998, Protein expression and purification.

[5]  J. Chirgwin,et al.  Solubility of proteins isolated from inclusion bodies is enhanced by fusion to maltose-binding protein or thioredoxin. , 1998, Protein expression and purification.

[6]  B. Leiting,et al.  High-level expression of soluble protein in Escherichia coli using a His6-tag and maltose-binding-protein double-affinity fusion system. , 1997, Protein expression and purification.

[7]  P. Alzari,et al.  The MBP fusion protein restores the activity of the first phosphatase domain of CD45 , 1997, FEBS letters.

[8]  T. Caldas,et al.  Chaperone Properties of the Bacterial Periplasmic Substrate-binding Proteins* , 1997, The Journal of Biological Chemistry.

[9]  V. de Lorenzo,et al.  Design of a solubilization pathway for recombinant polypeptides in vivo through processing of a bi-protein with a viral protease. , 1997, Protein engineering.

[10]  A. Fersht,et al.  A structural model for GroEL-polypeptide recognition. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[11]  Y. Kimata,et al.  A novel mutation which enhances the fluorescence of green fluorescent protein at high temperatures. , 1997, Biochemical and biophysical research communications.

[12]  Jim Haseloff,et al.  Mutations that suppress the thermosensitivity of green fluorescent protein , 1996, Current Biology.

[13]  C. D’Santos,et al.  Expression of recombinant rat myo-inositol 1,4,5-trisphosphate 3-kinase B suggests a regulatory role for its N-terminus. , 1996, The Biochemical journal.

[14]  S. Kain,et al.  Optimized codon usage and chromophore mutations provide enhanced sensitivity with the green fluorescent protein. , 1996, Nucleic acids research.

[15]  T. Oas,et al.  High-level misincorporation of lysine for arginine at AGA codons in a fusion protein expressed in Escherichia coli. , 1996, Journal of molecular biology.

[16]  J. Kollár,et al.  Purification and characterization of decorin core protein expressed in Escherichia coli as a maltose-binding protein fusion. , 1996, Analytical biochemistry.

[17]  J. Bodley,et al.  Expression, Purification, and Characterization of the G Domain ofSaccharomyces cerevisiaeElongation Factor 2 , 1996 .

[18]  R. Sutherland,et al.  High activity, soluble, bacterially expressed human vitamin D receptor and its ligand binding domain , 1996, Journal of cellular biochemistry.

[19]  J. Gouaux,et al.  Overexpression of bacterio‐opsin in Escherichia coli as a water‐soluble fusion to maltose binding protein: Efficient regeneration of the fusion protein and selective cleavage with trypsin , 1996, Protein science : a publication of the Protein Society.

[20]  W. Stemmer,et al.  Improved Green Fluorescent Protein by Molecular Evolution Using DNA Shuffling , 1996, Nature Biotechnology.

[21]  N. Warne,et al.  Histidine Patch Thioredoxins , 1996, The Journal of Biological Chemistry.

[22]  A. Blondel,et al.  Destabilizing interactions between the partners of a bifunctional fusion protein. , 1996, Protein engineering.

[23]  J. Bodley,et al.  Expression, purification, and characterization of the G domain of Saccharomyces cerevisiae elongation factor 2. , 1996, Protein expression and purification.

[24]  S Falkow,et al.  FACS-optimized mutants of the green fluorescent protein (GFP). , 1996, Gene.

[25]  W. Dougherty,et al.  Expression and purification of a recombinant tobacco etch virus NIa proteinase: biochemical analyses of the full-length and a naturally occurring truncated proteinase form. , 1995, Virology.

[26]  Y. Kashi,et al.  Residues in chaperonin GroEL required for polypeptide binding and release , 1994, Nature.

[27]  R. Williamson,et al.  Solution structure of the active domain of tissue inhibitor of metalloproteinases-2. A new member of the OB fold protein family. , 1994, Biochemistry.

[28]  H. Nikaido.  Maltose transport system of Escherichia coli: An ABC‐type transporter , 1994, FEBS letters.

[29]  M. Uhlén,et al.  Engineering proteins to facilitate bioprocessing. , 1994, Trends in biotechnology.

[30]  M. Uhlén,et al.  Enhanced in vitro refolding of insulin-like growth factor I using a solubilizing fusion partner. , 1994, Biochemistry.

[31]  L. Rosenberg,et al.  Rat liver mitochondrial processing peptidase. Both alpha- and beta-subunits are required for activity. , 1994, The Journal of biological chemistry.

[32]  G. Hannon,et al.  A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4 , 1993, Nature.

[33]  G. Volckaert,et al.  Carboxyl terminus is essential for intracellular folding of chloramphenicol acetyltransferase. , 1993, The Journal of biological chemistry.

[34]  J. Mccoy,et al.  A Thioredoxin Gene Fusion Expression System That Circumvents Inclusion Body Formation in the E. coli Cytoplasm , 1993, Bio/Technology.

[35]  K. Vousden.  Interactions of human papillomavirus transforming proteins with the products of tumor suppressor genes. , 1993, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[36]  N. Grindley,et al.  Binding of the IS903 transposase to its inverted repeat in vitro. , 1992, The EMBO journal.

[37]  M. Uhlén,et al.  Fusion proteins in biotechnology. , 1992, Current opinion in biotechnology.

[38]  M. J. Cormier,et al.  Primary structure of the Aequorea victoria green-fluorescent protein. , 1992, Gene.

[39]  F. Quiocho,et al.  The 2.3-A resolution structure of the maltose- or maltodextrin-binding protein, a primary receptor of bacterial active transport and chemotaxis. , 1992, The Journal of biological chemistry.

[40]  J. Louis,et al.  Autoprocessing of the HIV-1 protease using purified wild-type and mutated fusion proteins expressed at high levels in Escherichia coli. , 1991, European journal of biochemistry.

[41]  F. Quiocho,et al.  Progress in the identification of interaction sites on the periplasmic maltose binding protein from E coli. , 1990, Biochimie.

[42]  B. O’Malley,et al.  High level expression of a truncated chicken progesterone receptor in Escherichia coli. , 1990, The Journal of biological chemistry.

[43]  F. Studier,et al.  Use of T7 RNA polymerase to direct expression of cloned genes. , 1990, Methods in enzymology.

[44]  Catherine H. Schein,et al.  Production of Soluble Recombinant Proteins in Bacteria , 1989, Nature Biotechnology.

[45]  D. Ecker,et al.  Ubiquitin fusion augments the yield of cloned gene products in Escherichia coli. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[46]  C Marque,et al.  Human abdominal EHG processing for uterine contraction monitoring. , 1989, Biotechnology.

[47]  C. Schein,et al.  Formation of Soluble Recombinant Proteins in Escherichia Coli is Favored by Lower Growth Temperature , 1988, Bio/Technology.

[48]  M. Hofnung,et al.  Sequences of the malE gene and of its product, the maltose-binding protein of Escherichia coli K12. , 1984, The Journal of biological chemistry.

[49]  B Guss,et al.  Gene fusion vectors based on the gene for staphylococcal protein A. , 1983, Gene.

[50]  Jeffrey H. Miller.  Experiments in molecular genetics , 1972 .