Please use this identifier to cite or link to this item: http://hdl.handle.net/2440/11362
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Type: Journal article
Title: Introduction of spacer peptides N-terminal to a cleavage recognition motif in recombinant fusion proteins can improve site-specific cleavage
Author: Polyak, S.
Forsberg, G.
Forbes, B.
McNeil, K.
Aplin, S.
Wallace, J.
Citation: Protein Engineering, 1997; 10(6):615-619
Publisher: Oxford University Press
Issue Date: 1997
ISSN: 0269-2139
1460-213X
Statement of
Responsibility: 
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
RMID: 0030004367
DOI: 10.1093/protein/10.6.615
Appears in Collections:Biochemistry publications

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