High-Level Expression of Soluble Protein inEscherichia coliUsing a His6-Tag and Maltose-Binding-Protein Double-Affinity Fusion System

Pryor, KellyAnn D.; Leiting, Barbara

doi:10.1006/prep.1997.0759

Cited by 224 publications

(84 citation statements)

References 18 publications

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“…Most MBP-tagged proteins were primarily lost in the flow-through. A low binding efficiency of MBP-tagged proteins has been reported previously and is a result of the low affinity of MBP for its matrix (21).…”

Section: Ht Protein Purification Under Nondenaturing Conditionsmentioning

confidence: 65%

Proteome-scale purification of human proteins from bacteria

Braun

Shen

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

246

139

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The completion of the human genome project and the development of high-throughput approaches herald a dramatic acceleration in the pace of biological research. One of the most compelling next steps will be learning the functional roles of all proteins. Achievement of this goal depends in part on the rapid expression and isolation of proteins at large scale. We exploited recombinational cloning to facilitate the development of methods for the high-throughput purification of human proteins. cDNAs were introduced into a master vector from which they could be rapidly transferred into a variety of protein expression vectors for further analysis. A test set of 32 sequenceverified human cDNAs of various sizes and activities was moved into four different expression vectors encoding different affinity-purification tags. By means of an automatable 2-hr protein purification procedure, all 128 proteins were purified and subsequently characterized for yield, purity, and steps at which losses occurred. Under denaturing conditions when the His 6 tag was used, 84% of samples were purified. Under nondenaturing conditions, both the glutathione S-transferase and maltose-binding protein tags were successful in 81% of samples. The developed methods were applied to a larger set of 336 randomly selected cDNAs. Sixty percent of these proteins were successfully purified under denaturing conditions and 82% of these under nondenaturing conditions. A relational database, FLEXProt, was built to compare properties of proteins that were successfully purified and proteins that were not. We observed that some domains in the Pfam database were found almost exclusively in proteins that were successfully purified and thus may have predictive character.W ith the application of large-scale and high-throughput (HT) approaches to biological and medical questions, biology has embraced a new era of technology development and information collection. The great task lying ahead is to elucidate the functions of all proteins encoded in the genomes of sequenced model organisms. This process involves collection of information about the temporal, spatial, and physiological regulation of proteins, their interaction partners, biochemical activities, posttranslational modifications, and the mutual influence of all these parameters on the physiology of the organism. Over the past several decades, biologists and biochemists have amassed a large collection of powerful tools for the study of individual proteins. However, compared with the study of nucleic acids, the HT study of proteins is still in its infancy. The next great challenge in biology will be to adapt these tools and develop new ones that enable the simultaneous and parallel study of thousands of proteins.The elucidation of biochemical activity and protein-protein interactions are central aspects of understanding protein function. Protein microarrays provide one platform for biochemical experiments to be carried out at extraordinary pace (1-3). However, this exciting technology calls attention to the quest...

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Section: Ht Protein Purification Under Nondenaturing Conditionsmentioning

confidence: 65%

Proteome-scale purification of human proteins from bacteria

Braun

Shen

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

246

139

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“…The production yields we report, 70-120 mg of puri®ed MBP-scFv from 2 g of wet bacterial paste exceed the best periplasmically expressed scFv 78 and parallels the production level we achieve from insoluble cytoplasmic inclusion bodies by matrix-assisted refolding. 79 It is conceivable that, with further optimization of growth and induction conditions (medium composition, inducer concentration, growth temperature), 7,42 our system can be made even more ef®cient. In addition, as we show here, certain scFvs that are very insoluble when expressed in bacteria, such as the anti-¯uorescein scFv 4-4-20/212, can be produced as a soluble MBPscFv with very little effort ( Figure 6).…”

Section: Discussionmentioning

confidence: 99%

Escherichia coli maltose-binding protein as a molecular chaperone for recombinant intracellular cytoplasmic single-chain antibodies 1 1Edited by R. Huber

Bach

Mazor

Shaky

et al. 2001

Journal of Molecular Biology

166

119

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Recombinant single-chain antibodies (scFvs) that are expressed in the cytoplasm of cells are of considerable biotechnological and therapeutic potential. However, the reducing environment of the cytoplasm inhibits the formation of the intradomain disul®de bonds that are essential for correct folding and functionality of these antibody fragments. Thus, scFvs expressed in the cytoplasm are mostly insoluble and inactive.Here, we describe a general approach for stabilizing scFvs for ef®cient functional expression in the cell cytoplasm in a soluble, active form. The scFvs are expressed as C-terminal fusions with the Escherichia coli maltose-binding protein (MBP). We tested a large panel of scFvs that were derived from hybridomas and from murine and human scFv phage display and expression libraries by comparing their stability and functionality as un-fused versus MBP fused proteins. We found that MBP fused scFvs are expressed at high levels in the cytoplasm of E. coli as soluble and active proteins regardless of the redox state of the bacterial cytoplasm. In contrast, most un-fused scFvs can be produced (to much lower levels) in a functional form only when expressed in trxB À but not in trxB E. coli cells. We show that MBP-scFv fusions are more stable than the corresponding un-fused scFvs, and that they perform more ef®ciently in vivo as cytoplasmic intrabodies in E. coli. Thus, MBP seems to function as a molecular chaperone that promotes the solubility and stability of scFvs that are fused to it.

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“…coli MBP is an excellent solubility enhancer [3,4,5,6], but it is not the most effective affinity tag from the standpoint of protein purification [7,8]. Some fusion proteins do not bind efficiently to amylose resin, and even when they do, the purity of proteins after amylose affinity chromatography is usually inadequate.…”

Section: Introductionmentioning

confidence: 99%

Mutations that alter the equilibrium between open and closed conformations of Escherichia coli maltose-binding protein impede its ability to enhance the solubility of passenger proteins

Nallamsetty

Waugh

2007

Biochemical and Biophysical Research Communications

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Certain highly soluble proteins, such as Escherichia coli maltose-binding protein (MBP), have the ability to enhance the solubility of their fusion partners, making them attractive vehicles for the production of recombinant proteins, yet the mechanism of solubility enhancement remains poorly understood. Here, we report that the solubility-enhancing properties of MBP are dramatically affected by amino acid substitutions that alter the equilibrium between its "open" and "closed" conformations. Our findings indicate that the solubility-enhancing activity of MBP is mediated by its open conformation and point to a likely role for the ligand-binding cleft in the mechanism of solubility enhancement.Keywords maltose binding protein; solubility enhancer; affinity tag; fusion protein; solubility tag Proteins that normally accumulate in the form of insoluble aggregates when they are overproduced in E. coli can sometimes be recovered in a soluble, properly folded form if they are fused to a solubility-enhancing partner [1]. Consequently, the use of solubility-enhancing fusion partners has become an attractive alternative to the refolding of proteins. Many proteins have been reported to exhibit solubility-enhancing characteristics, including MBP, NusA, DsbA, thioredoxin, T7PK, Skp, SUMO, GB1 and the ZZ domain [2], although the evidence supporting these claims is stronger in some instances than in others. Yet the mechanism(s) by which certain soluble proteins enhance the solubility of their fusion partners remains poorly understood.E. coli MBP is an excellent solubility enhancer [3,4,5,6], but it is not the most effective affinity tag from the standpoint of protein purification [7,8]. Some fusion proteins do not bind efficiently to amylose resin, and even when they do, the purity of proteins after amylose affinity chromatography is usually inadequate. Two groups recently described mutations in MBP that increase its affinity for maltose [9,10]. Using a variety of experimental techniques, both groups reached the conclusion that these mutations exert their effect by altering the equilibrium between the "open" and "closed" conformations of MBP so as to favor the latter. In the open conformation, the ligand-binding cleft of MBP is exposed to solvent, whereas the closed conformation resembles that of the ligand-bound protein in which the cleft is largely buried.* Corresponding author, David S. Waugh, Ph.D., Tel: (301) 846-1842, Fax: (301) 846-7148, Email: waughd@ncifcrf.gov. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. We originally set out to determine whether these mutations wou...

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High-Level Expression of Soluble Protein inEscherichia coliUsing a His6-Tag and Maltose-Binding-Protein Double-Affinity Fusion System

Cited by 224 publications

References 18 publications

Proteome-scale purification of human proteins from bacteria

Proteome-scale purification of human proteins from bacteria

Escherichia coli maltose-binding protein as a molecular chaperone for recombinant intracellular cytoplasmic single-chain antibodies 1 1Edited by R. Huber

Mutations that alter the equilibrium between open and closed conformations of Escherichia coli maltose-binding protein impede its ability to enhance the solubility of passenger proteins

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