Construction of Protease Resistant Mutein of Human CNTF and Its Expression in Pichia pastoris
蛋白酶抗性人睫状神经营养因子突变体基因的构建及其在巴斯德毕赤酵母中的表达

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.