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

Construction of Protease Resistant Mutein of Human CNTF and Its Expression in Pichia pastoris

AX15是一种比天然睫状神经营养因子具有更高的生物学活性、更好的稳定性和可溶性的hCNTF突变体。在巴斯德毕赤酵母中表达时AX15易发生降解。氨基酸序列分析表明降解位于由 12和 13位氨基酸残基组成的双碱性氨基酸之后。根据KEX2蛋白酶的底物专一性把双碱性氨基酸从RR变为RK ,构建了KEX2抗性的AX15突变体AX15 (R13K)。AX15 (R13K)的稳定性得到了显著的提高 ,在诱导 10 0h后也未发生降解。利用超滤浓缩和凝胶过滤得到了纯度 >90 %的AX15 (R13K)。TF 1细胞存活实验表明AX15 (R13K)具有与AX15相同的生物学活性。蛋白酶抗性人睫状神经营养因子突变体可能具有更好的体内稳定性 ,在临床应用上具有潜在的优势。

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.