Introduction of spacer peptides N-terminal to a cleavage recognition motif in recombinant fusion proteins can improve site-specific cleavage.
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.
Polyak
S W
SW
Co-operative Research Centre for Tissue Growth and Repair, Department of Biochemistry, University of Adelaide, Australia.
Forsberg
G
G
Forbes
B E
BE
McNeil
K A
KA
Aplin
S E
SE
Wallace
J C
JC
D000595:Amino Acid Sequence; D000818:Animals; D013006:Growth Hormone / Q000138:chemical synthesis / Q000737:chemistry / Q000302:isolation & purification; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D021382:Protein Sorting Signals / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis* / Q000737:chemistry* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000378:metabolism; D013381:Subtilisins / Q000235:genetics / Q000378:metabolism; D013552:Swine
D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D007335:Insulin-Like Growth Factor II; D013006:Growth Hormone; D013381:Subtilisins