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.
Department of Food Science, National Taiwan Ocean University, Keelung, Taiwan 202. sjiang@mail.ntou.edu.tw
Shann-Tzong
ST
Jiang
Gen-Hung
GH
Chen
Shye-Jye
SJ
Tang
Ching-San
CS
Chen
D000818:Animals; D002645:Chickens / Q000235:genetics*; D015891:Cystatins / Q000737:chemistry* / Q000235:genetics*; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D005615:Freezing; D015870:Gene Expression; D006031:Glycosylation; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins
D015891:Cystatins; D011994:Recombinant Proteins