抗菌肽DCD-1L的随机突变及在毕赤酵母中的表达

Random Mutation of Antibacterial Peptide DCD-1L and Expression in Pichia pastoris

目的用毕赤酵母构建抗菌肽DCD-1L的随机多肽库,并使随机多肽在培养基中表达。方法用定向进化的方法在基因内部造成随机突变,然后将其构建到含有强启动子GAP和α-factor信号肽序列的巴斯德毕赤酵母载体pGAPZαC中,线性化后转化到巴斯德毕赤酵母蛋白酶缺陷株SMD1168中,形成突变库。利用抗生素Zeocin筛选出阳性克隆,并用SDS-PAGE鉴定表达蛋白质。结果肽库库容达到7.8×103个cfu,在上清中鉴定出了表达蛋白。结论成功构建了抗菌肽DCD-1L的随机多肽库,并实现了在培养基上清中的表达。

Objective To construct the mutation library of antibacterial peptide DCD-1L and achieve the expression in the medium. Methods The genes with the sequence of random mutations through splicing by overlap extension were inserted into the Pichia pastoris expression vector pGAPZαC which contained GAP promotor and α-factor signal sequence. Then, the pGAPZαC was transferred into a protease-defective strain of Pichia pastoris SMD 1168, and a mutation library was formed. The positive clone strains were screened with Zeocin and the protein expression was identified by SDS-PAGE. Results The storage capacity of about 7.8 × 10^3 cfu was achieved and the protein expression was found in the supernatant. Conclusion The mutation library of antibacterial peptide DCD-1L can be established and the protein expression can be achieved in the supernatant.