摘要
A cDNA library was constructed with 1.5×10~6 pfu from rice immature seeds,fromwhich a cDNA clone for rice thiol proteinase inhibitor,oryzacystatin(OC),was isolated byscreening with synthesized oligodeoxynucleotide probe,which contained a 309bp open read-ing frame,84bp 5′-end noncoding region and a poly(A)signal AATAAA at the 3′-end fol-lowed by 31Nt poly(A).Then the coding region of OC was amplified and inserted into thedownstream of λP_RP_L promoter for thermal-inducible expression in E.coli.Shifting the cul-ture temperature from 30℃ to 42℃ led to a high level expression of OC,which exhibited adistinct band of 12.0 kDa and accounted for at least 10% of the total soluble proteins fromSDS-PAGE.The papain-inhibitory activity of the expressed OC was further confirmed.
A cDNA library was constructed with 1.5×10~6 pfu from rice immature seeds,from which a cDNA clone for rice thiol proteinase inhibitor,oryzacystatin(OC),was isolated by screening with synthesized oligodeoxynucleotide probe,which contained a 309bp open read- ing frame,84bp 5′-end noncoding region and a poly(A)signal AATAAA at the 3′-end fol- lowed by 31Nt poly(A).Then the coding region of OC was amplified and inserted into the downstream of λP_RP_L promoter for thermal-inducible expression in E.coli.Shifting the cul- ture temperature from 30℃ to 42℃ led to a high level expression of OC,which exhibited a distinct band of 12.0 kDa and accounted for at least 10% of the total soluble proteins from SDS-PAGE.The papain-inhibitory activity of the expressed OC was further confirmed.
基金
Supported by Prime Minister FouNdation
the High Techriology Research and Development Programme of China
Rockefeller Foun-dation.
