扩展青霉碱性脂肪酶cDNA的克隆和表达

Cloning and Expression of cDNA Encoding Alkaline Lipase from Penicillium expansum

采用 ＴＲＩｚｏｌ试剂一步法所抽提的总 ＲＮＡ的 Ｄ（２６０）／Ｄ（２８０）值为 １.８２，甲醛变性电泳呈现真核微生物所特有的 ２８Ｓ ｒＲＮＡ和 １８Ｓ ｒＲＮＡ 条带．根据扩展青霉所产碱性脂肪酶（Ｌｉｐ ＰＥ）Ｎ末端 ２０个氨基酸残基序列和真核生物 ｍＲＮＡ３’端具有 ｐｏｌｙ（Ａ）等所提供的生物信息，采用 ＲＴ－ＰＣＲ技术和 ３’－ＲＡＣＥ法扩增了 ＬｉｐＰＥ成熟肽编码区和３’非纪码区的ｃＤＮＡ，直接将该ＰＣＲ产物克隆至ｐＵＣｍ－Ｔ载体中。序列分析表明，该碱性脂肪酶含有２５８个氨基酸，其中保守的五肽序列为 Ｇｌｙ－Ｈｉｓ－Ｓｅｒ－Ｌｅｕ－Ｇｌｙ。进一步采用 ＣｌｏｎＴｅｃｈ公司的 ＳＭＡＲＴ～（ＴＭ）ＰＣＲ ｃＤＮＡ文库构建试剂盒，扩增、克隆和测定了自转录起始点至纪码区的 ｃＤＮＡ片段，从而完成了 Ｌｉｐ ＥＰ完整 ｃＤＮＡ的分析测定．最后将编码完整脂肪酶蛋白的ｃＤＮＡ克隆至 ｐＧＥＸ－５Ｘ－３表达载体中．在大肠杆菌 ＢＬ２１中进行 ＩＰＴＧ诱导表达，ＳＤＳ－ＰＡＧＥ检测结果表明，所表达的 ＧＳＴ－Ｌｉｐ ＥＰ融合蛋白分子质量约为 ５３ｋｕ；

Total RNA was isolated from Penicillium expansum WMC20718 with TRIzol reagent by one step method, by which the ratio of D(260) to D(280) was 1.82, and 28S and 18S rRNA bands characterized as fungal microbes of isolated total RNA on formaldehyde denatured electrophoresis were presented. With the information of N- terminal amino acid sequence of Lip PE and 3'- terminal poly (A) of eukaryotic mRNA, the mature peptide encoding region with 3' non-encoding one of Lip PE cDNA from WMC20718 was amplified with RT-PCR, and PCR product was cloned into the vector pUCm-T. Sequence analysis showed that the mature protein of Lip PE was composed of 258 amino acids, in which a well-conserved -Gly-His-Ser-Leu-Gly- sequence existed. Amplification, cloning and sequencing of eDNA fragment from the transcription start point of mRNA to Lip PE encoding region were also carried out using SMARTTM PCR eDNA Synthesis Kit (Clon Tech). From above two steps of PCR, an entire Lip PE eDNA sequence from WMC20718 was determined and analyzed. The cDNA encoding entire Lip PE mature peptide was cloned into ex- pression plasmid pGEX-5X-3 and expressed in E. coli BL21 by IPTG induction. SDS-PAGE analysis for the expression product of pGEX-Lip PE in F. coli showed that the molecular mess of the expressed fusion protein is about 53 ku. Western blot analysis confirmed that the cloned cDNA from WMC20718 was the gene encoding mature Lip PE.