Effect of glycosylation modification (N-Q-(108)I --> N-Q-(108)T) on the freezing stability of recombinant chicken Cystatin overexpressed in Pichia pastoris X-33

The cDNAs encoding chicken cystatin and its N-glycosylation-modified mutant (Asn(106)-Ile(108)-->Asn(106)-Thr(108)) were cloned into the pGAPZ alpha C expression vector, using the GAP as promoter and Zeocin as resistant agent, and transformed into Pichia pastoris X-33 expression host. The effect of N-glycosylation on the stability of recombinant chicken cystatin was investigated. A large quantity of recombinant chicken cystatin and the Asn(106)-glycosylated cystatins were expressed and secreted into broth using alpha-factor preprosequence. The K(i) of the recombinant chicken cystatin (0.08 nM) was similar to that of wild-type chicken cystatin (0.05 nM). They acted as a competitive inhibition reaction against papain. According to the K(i), the inhibition ability of Asn(106)-glycosylated mutant cystatin (K(i) = 9.5 nM) was weaker than that of the wild-type one. However, N-glycosylation at Asn(106) substantially enhanced the freezing stability of recombinant chicken cystatin overexpressed in P. pastoris.