Engineering soluble proteins for structural genomics

Pédelacq, J.D.; Piltch, Emily; Liong, E.C.; Berendzen, Joel; Kim, Chang-Yub; Rho, Beom-Seop; Park, Min S; Terwilliger, Thomas C.; Waldo, Geoffrey S.

doi:10.1038/nbt732

Cited by 168 publications

(132 citation statements)

References 33 publications

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“…Concerning the engineering of enzymes toward novel desired properties, like altered substrate specificity and improved activity, solubility, and stability; researchers have relied on the use of rational design and/or directed-evolution methods in combination with appropriate screening and selection procedures (1,10,11,22). For instance, various mutagenesis procedures and subsequent screening via assays that report on the successful folding of a protein of interest (9, 32, 45) have been used to engineer protein variants exhibiting improved solubility (35,37,46). Despite the obvious success of using such folding reporters in solubility/folding engineering projects, there is a risk that the engineered protein may lose its inherent activity since these screening procedures in general do not select for retained activity but only improved solubility/folding.…”

mentioning

confidence: 99%

Novel Fluorescence-Assisted Whole-Cell Assay for Engineering and Characterization of Proteases and Their Substrates

Kostallas

Samuelson

2010

Appl Environ Microbiol

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We have developed a sensitive and highly efficient whole-cell methodology for quantitative analysis and screening of protease activity in vivo. The method is based on the ability of a genetically encoded protease to rescue a coexpressed short-lived fluorescent substrate reporter from cytoplasmic degradation and thereby confer increased whole-cell fluorescence in proportion to the protease's apparent activity in the Escherichia coli cytoplasm. We demonstrated that this system can reveal differences in the efficiency with which tobacco etch virus (TEV) protease processes different substrate peptides. In addition, when analyzing E. coli cells expressing TEV protease variants that differed in terms of their in vivo solubility, cells containing the most-soluble protease variant exhibited the highest fluorescence intensity. Furthermore, flow cytometry screening allowed for enrichment and subsequent identification of an optimal substrate peptide and protease variant from a large excess of cells expressing suboptimal variants (1:100,000). Two rounds of cell sorting resulted in a 69,000-fold enrichment and a 22,000-fold enrichment of the superior substrate peptide and protease variant, respectively. Our approach presents a new promising path forward for high-throughput substrate profiling of proteases, engineering of novel protease variants with desired properties (e.g., altered substrate specificity and improved solubility and activity), and identification of protease inhibitors.Proteases constitute a group of enzymes that irreversibly catalyze the cleavage of peptide bonds and represent approximately 2% of all protein-encoding genes in living organisms (39). Besides acting as virulence factors for many pathogens (16), proteases are crucial for the regulation of numerous biological processes that influence the life and death of a cell (4). These enzymes also underlie several pathological conditions, such as cancer (13) and neurodegenerative (20) and cardiovascular (8) diseases. A key issue for increasing our knowledge about such complex biological processes, and thereby hopefully also providing possibilities for new therapeutic strategies, is to deduce the proteases' substrate repertoires. Consequently, a lot of efforts around the world are dedicated to the characterization of proteases and their substrates (2, 31). In addition to their biological importance, proteases have attracted much interest in several biotechnological and industrial applications, such as removal of "fusion tags" from recombinant target proteins (38), as supplements in dishwashing and laundry detergents, or for bating of hides and skin in the leather industry (41, 44). Sometimes, however, their use is hindered due to limitations inherent to a specific protease: for example, low solubility, poor enzyme stability and specificity, or limited activity. It would therefore be of great aid to have powerful and straightforward methods available that facilitate the engineering of novel protease variants not suffering from such limitations.Traditionally, pr...

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confidence: 99%

Novel Fluorescence-Assisted Whole-Cell Assay for Engineering and Characterization of Proteases and Their Substrates

Kostallas

Samuelson

2010

Appl Environ Microbiol

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“…To tackle these complications attributed by E. coli expressions system number of animal and plant expression system have designed [37,38]. Though these engineered expression system have made significant contribution for expression of eukaryotic proteins often failed in E. coli system but again failed to design the simple and efficient, high-yield, low-cost methods which can be used for the production of proteins in large amount [39].…”

Section: Complications With Conventional Expression Systemmentioning

confidence: 99%

New Generation Expression Host System- Aiming high for commercial production of recombinant protein

Verma¹

2012

IOSJPBS

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“…[3][4][5][6][7] Proteins expressed as inclusion bodies were denatured, purified, and refolded to yield soluble forms. 8,9 Several proteins showed enhanced solubility and long-term stability by simultaneous addition of charged amino acids L-Arg and L-Glu at 50 mM to the buffer, which increased the maximal concentration up to 8.7 times.…”

Section: 4mentioning

confidence: 99%

Solution Structure of Water-soluble Mutant of Crambin and Implication for Protein Solubility

Kang¹,

Lim²,

Lee³

et al. 2011

Bulletin of the Korean Chemical Society

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Water-soluble mutant of intrinsically insoluble protein, crambin, was produced by mutagenesis based on the sequence analysis with homologous proteins. Thr1, Phe13, and Lys33 of crambin were substituted for Lys, Tyr, and Lys, respectively. The resultant mutant was soluble in aqueous buffer as well as in dodecylphosphocholine (DPC) micelle solution. The H- 15N spectrum of the mutant crambin showed spectral similarity to that of the wild-type protein except for local regions proximal to the sites of mutation. Solution structure of water-soluble mutant crambin was determined in aqueous buffer by NMR spectroscopy. The structure was almost identical to the wild-type structure determined in non-aqueous solvent. Subtle difference in structure was very local and related to the change of the intra-and inter-protein hydrophobic interaction of crambin. The structural details for the enhanced solubility of crambin in aqueous solvent by the mutation were provided and discussed.

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Engineering soluble proteins for structural genomics

Cited by 168 publications

References 33 publications

Novel Fluorescence-Assisted Whole-Cell Assay for Engineering and Characterization of Proteases and Their Substrates

Novel Fluorescence-Assisted Whole-Cell Assay for Engineering and Characterization of Proteases and Their Substrates

New Generation Expression Host System- Aiming high for commercial production of recombinant protein

Solution Structure of Water-soluble Mutant of Crambin and Implication for Protein Solubility

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