Recombinant proteins specific cleavage

Table 2 Approaches for specific cleavage of recombinant proteins...

Figure 2 A general overexpresion vector. Most Escherichia coii vectors for recombinant protein production will have most of these features. The N- and C-terminal fusions and associated protease cleavage sites are optional. The multiple cloning site (MCS) may be replaced by DNA sequences for site-specific recombination in some vectors.

TEV protease has become one of the most popular proteases for use in separating recombinant proteins from their attached fusion partners because of its stringent sequence specificity and activity over a broad temperature range. The protein can be recombinantly produced in E. coli with a His tag for easy purification and subsequent removal from reaction mixtures.290 304 305 The wild-type enzyme suffers from autoproteolysis, which leads to a truncated form with vastly reduced activity therefore, specific mutants are used that stabilize the protein and increase its catalytic efficiency.288 The enzyme is well studied and its crystal structure has been solved.287 289 306 An additional benefit of TEV protease is that many amino acids are tolerated in the PI position of its recognition site however, efficiency decreases for some amino acids and proline abolishes cleavage.287 Since the PI amino acid will remain on the protein of interest when N-terminal fusions are cleaved off, this does allow some flexibility to match the naturally occurring N-terminus post cleavage. 

The N-terminal fragment 1-62 and the C-terminal fragment 76-174 of two recombinant analogues of GCSF were isolated after enzymatic cleavage with the Lys-specific protease from Achromobacter lyticus. In one fragment, the only Lys in the protein was at position 62, and was immediately followed by Ser. In the other fragment, the only Lys, also followed by a... 

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