Construction of protease resistant mutein of human CNTF and its expression in Pichia pastoris / 生物工程学报
Chinese Journal of Biotechnology
;
(12): 394-397, 2004.
Article
in Chinese
| WPRIM
| ID: wpr-249975
ABSTRACT
AX15 is a mutein of naturally occurring human ciliary neurophic factor (hCNTF), with improved biological activity, stability and solubility. AX15 is susceptible to protease degradation when expressed in Pichia pastoris. Amino acid sequencing revealed the degradation was occurred behind position 12 and 13 amino acid residues, which constitute a dibasic site, RR. Based on the substrate specificity of KEX2, a KEX2 resistant mutein of AX15-AX15 (R13K) was constructed, in which RR was replaced by RK. It was demonstrated that the stability of AX15 (R13K) improved significantly, as no degradation was detected even after 120 hours of induction. AX15 (R13K) was purified to homogeneity by ultrafiltration and gel filtration. TF-1 cell survival bioassay showed AX15 (R13K) had equivalent specific activity to AX15. The protease resistant mutein of AX15 may have greater in vivo stability and thus have superior therapeutic potential.
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Peptide Hydrolases
/
Pichia
/
Recombinant Proteins
/
Chemistry
/
Ciliary Neurotrophic Factor
/
Mutant Proteins
/
Genetic Vectors
/
Genetics
/
Metabolism
/
Mutation
Limits:
Humans
Language:
Chinese
Journal:
Chinese Journal of Biotechnology
Year:
2004
Type:
Article
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