[Construction of protease resistant mutein of human CNTF and its expression in Pichia pastoris].
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.
Zhao
Hong-Liang
HL
Beijing Institute of Biotechnology, Beijing 100071, China. hlzhao@vip.sina.com
Xue
Chong
C
Xiong
Xiang-Hua
XH
Zhang
Wei
W
Zhu
Hou-Chu
HC
Liu
Zhi-Min
ZM
D020934:Ciliary Neurotrophic Factor / Q000096:biosynthesis / Q000235:genetics*; D005822:Genetic Vectors; D006801:Humans; D050505:Mutant Proteins / Q000096:biosynthesis* / Q000235:genetics; D009154:Mutation; D010447:Peptide Hydrolases / Q000737:chemistry*; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D020934:Ciliary Neurotrophic Factor; D050505:Mutant Proteins; D011994:Recombinant Proteins; D010447:Peptide Hydrolases