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|Title:||Introduction of spacer peptides N-terminal to a cleavage recognition motif in recombinant fusion proteins can improve site-specific cleavage|
|Citation:||Protein Engineering, 1997; 10(6):615-619|
|Publisher:||Oxford University Press|
|Steve W Polyak, Göran Forsberg, Briony E Forbes, Kerrie A McNeil, Sally E Aplin, and John C Wallace|
|Abstract:||To improve site-specific cleavage of a methionyl porcine growth hormone [[Met1]-pGH(1-46)-IGF-II] fusion protein by the enzyme H64A subtilisin, a series of flexible, unstructured spacer peptides were introduced N-terminal to the cleavage site. When enzymatic digestion preceded refolding of the fusion proteins, IGF-II could only be liberated from substrates which contained spacer peptides. Compared with the parent construct, the yield of IGF-II from refolded fusion proteins containing spacers was improved up to two-fold. Furthermore, this cleavage rate was improved by removing a competing protease recognition motif from the fusion partner. These data show that fusion partners can influence site-specific proteolysis of fusion proteins. Introduction of flexible spacers between the moieties can alleviate these interactions.|
|Keywords:||Animals; Swine; Humans; Growth Hormone; Subtilisins; Insulin-Like Growth Factor II; Protein Sorting Signals; Recombinant Proteins; Recombinant Fusion Proteins; Mutagenesis, Site-Directed; Amino Acid Sequence; Protein Folding; Molecular Sequence Data|
|Appears in Collections:||Biochemistry publications|
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