The acidity of protein fusion partners predominantly determines the efficacy to improve the solubility of the target proteins expressed in Escherichia coli

Su, Yu-Ching; Zou, Zhurong; Shen, Feng; Zhou, Pei; Cao, Lijuan

doi:10.1016/j.jbiotec.2007.01.015

Cited by 31 publications

(22 citation statements)

References 47 publications

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“…For instance, the highly acidic, solubility-enhancing SET tag [23] has been proposed to exert its effect by inhibiting aggregation through electrostatic repulsion and may not adopt a globular conformation at all. Su et al observed that globular proteins with increasingly higher net acidity exhibited a greater capacity to enhance the solubility of two passenger proteins [24]. However, in a comparative study of MBPs from a variety of microorganisms, no correlation between net charge and solubility-enhancing ability was observed [18].…”

Section: Discussionmentioning

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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Section: Discussionmentioning

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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“…35 In addition, the net charge of small and large tags is also important for their solubilizing ability. 36,37 It was suggested that mechanistically the electrostatic repulsions between proteins could prevent protein aggregation. 38 Moreover, the steric hindrance Would there be another type of chaperones inside the cells?…”

Section: Charge and Steric Hindrance As Important Factors For Stabilimentioning

confidence: 99%

RNA-mediated chaperone type for de novo protein folding

Choi¹,

Ryu²,

Seong³

2009

RNA Biology

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“…Two parameters NGPS%, DE-KR% from the solubility model of Wilkinson and Harrison (1991), the isoelectric point (pI) and the net charge at pH 7.0, can be regarded as the mirrors of the protein acidity. The fusion partner with higher acidity demonstrated a more significant efficacy in promoting the solubility of the target protein (Su et al, 2007). According to this model, EspA may be candidate of solubility enhancer due to its many properties, including high acidity, moderate hydrophilicity, and comparative low value of NGPS%.…”

Section: Discussionmentioning

confidence: 94%

“…In addition, several other fusion partners such as Trx, NusA, DsbA and IF2 have been employed to promote the solubility and stability of target proteins (Davis et al, 1999;LaVallie et al, 2000;Sorensen et al, 2003;Xu et al, 2007;Yasukawa et al, 1995;Zhang et al, 1998). Recently, the monomeric mutant of the Ocr protein (0.3 gene product) of bacteriophage T7 (Mocr) and the E. coli proteins Skp and Msb, have been used as fusion partners to enhance the solubility of passenger proteins DelProposto et al, 2009;Su et al, 2007). Many obstacles are encountered, however, when using these fusion partners.…”

Section: Introductionmentioning

confidence: 99%

EspA is a novel fusion partner for expression of foreign proteins in Escherichia coli

Cheng

Wang

et al. 2010

Journal of Biotechnology

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The acidity of protein fusion partners predominantly determines the efficacy to improve the solubility of the target proteins expressed in Escherichia coli

Cited by 31 publications

References 47 publications

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

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

RNA-mediated chaperone type for de novo protein folding

EspA is a novel fusion partner for expression of foreign proteins in Escherichia coli

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