文献类型：期刊文献

中文题名：信号肽疏水性的提高促进青霉素G酰化酶分泌

英文题名：Increase in Hydrophobicity of Signal Peptide Enhances Secretion of Penicillin G Acylase

作者：杨运桂[1];徐京宁[2];胡泰山[1];钱友存[1];杨胜利[1];龚毅[1]

机构：[1]中国科学院上海生命科学研究院上海生物工程研究中心,上海200233;[2]华东理工大学国家生物反应器工程重点实验室,上海200237

年份：2000

卷号：32

期号：2

起止页码：163

中文期刊名：生物化学与生物物理学报

收录：CSTPCD;;Scopus;CSCD:【CSCD2011_2012】；PubMed;

基金：国家"8 6 3"生物技术资助项目!No .10 2 11 0 2 0 1&&

语种：中文

中文关键词：青霉素G酰化酶;信号肽;疏水核心;疏水性;分泌

外文关键词：Penicillin G acylase; signal peptide; hydrophobic core; hydrophobicity

摘要：设计和合成了一段具有连续 10个亮氨酸强疏水核心的信号肽 (artificialsignalpeptide ,ASP) ,由EcoRI KpnI位点融合到青霉素G酰化酶 ( penicillinGacylase ,PAC)信号肽 (wildtypesignalpeptide,WTSP)的 - 4Pro位点 ,分别构建了PAC表达质粒 :pKKpacΔSP ,pKKpacWTSP ,pKKpacASP ,pETpacWTSP ,pETpacASP。ASP与WTSP均为 2 6个氨基酸 ,两者N端为MK ,C端均为PAVA。通过酶活性的测定及Western印迹分析 ,分别在tac,T7及氧调控表达系统中研究ASP和WTSP对PAC分泌的影响。结果表明 ,与WTSP相比 ,ASP对PAC酶活水平平均提高了约 63 % ,对PAC前体的分泌能力平均提高了约 4 1% ,通过提高信号肽疏水核心疏水性有效促进了青霉素G酰化酶的分泌。
An artificial strong hydrophobic signal peptide (ASP), containing ten leucines in tandem in the hydrophobic core, was utilized to replace the wild type signal peptide (WTSP) of penicillin G acylase (PAC), by the fusion to its -4 pro site. PAC expression plasmids, including pKKpacΔSP, pKKpacWTSP, pKKpacASP, pETpacWTSP and pETpacASP, were constructed. The length and the amino and carboxyl terminus amino acid composition of ASP and WTSP were kept identical. The activity assay and Western blotting analysis were used to study the effect of ASP and WTSP on the secretion of PAC in tac, T7 and dissolved oxygen regulation expression systems, respectively. Lack of signal peptide in pKKpacΔSP resulted in the accumulation of 91 kD PAC precursor (without signal peptide, but with the space peptide between α subunit and β subunit) in the cytosol, indicating that the secretion of PAC depends on the signal peptide. In BL21(pKKpacASP) cells, the PAC activity and proprecursor (with signal peptide and space peptide) processing capacity were increased by about 54% and 38.5%, respectively, in comparison with BL21(pKKpacWTSP) cells. Compared with BL21(DE3) (pETpacWTSP), however, the PAC activity and proprecursor processing capacity in BL21(DE3) (pETpacASP) were enhanced by about 69% and 43.5%, respectively. The PAC activity expressed from pETpacASP was about 67% more than that from pETpacWTSP in the dissolved oxygen regulated expression system GJ100. Resulting from the strong hydrophobicity of ASP, therefore, the PAC activity and proprecursor processing capacity were increased by about 63% and 41% on average, respectively, in comparison with WTSP. In conclusion, the increase in hydrophobicity of the signal peptide hydrophobic core enhanced the secretion of penicillin G acylase.