普通变形杆菌位置非特异性脂肪酶基因的克隆及在大肠杆菌中的表达

Cloning and expression of non-position-specific lipase gene from Proteus vulgaris

摘要:

摘要：【目的】本文拟克隆普通变形杆菌（Proteus vulgaris）脂肪酶基因，并实现其在大肠杆菌中的高效表达，并检验外源表达脂肪酶的催化性质。【方法】通过PCR方法，从P. vulgaris基因组中扩增其脂肪酶基因（PVL），并将其开放读码框区域连接到表达载体pET-DsbA及pMBP-P上，在大肠杆菌中用IPTG诱导表达。我们对培养基组分及培养条件进行优化，以获得最高的酶产量。用Ni柱亲和层析法对所得重组脂肪酶进行纯化，并考察其酶学性质。【结果】PVL基因编码区含864个碱基，编码含287个氨基酸的酶蛋白。该序列在GenBank的登录号为FJ643627。PVL基因在大肠杆菌BL21内诱导表达的最佳条件为：在pH8.5的LB培养基中添加15 mg/mL葡萄糖及200 μg/mL氨苄青霉素，在培养至OD600为1.2时加入100 μg/mL IPTG，15 ℃诱导15 h，最高酶活达到192.2 U/mL。通过亲和层析纯化了重组脂肪酶，得到一个约31 kDa的蛋白条带。外源表达的脂肪酶的催化特性与野生菌脂肪酶相似，具有催化的位置非特异性，对长链脂肪酸酯亲和性最高。【结论】PVL基因在大肠杆菌中的高效表达为P. vulgaris脂肪酶的进一步研究与应用奠定基础。

Abstract:

Abstract: [ Objective ] To produce Proteus vulgaris lipase (PVL) in large quantities, we cloned and expressed the lipase gene in Escherichia coli. [ Methods ] We cloned PVL gene by PCR method and then inserted the open reading frame of PVL gene into pET-DsbA and pMBP-P vectors. PVL gene was expressed in E. coli with the introduction of isopropyl β-D-1-thiogalactopyranoside (IPTG). We also studied the optimal culture conditions, including the concentrations of glucose, IPTG and ampicillin, induction temperature, and pH value of the medium. The characteristics of recombinant lipase were examined after affinitive purification by His-chelating affinity chromatography. [ Results ] The open reading frame of PVL gene consisted of 864 base pairs, encoding a protein of 287 amino acids. The sequence was deposited to GenBank with the accession number FJ643627. The gene was expressed in E. coli and active lipase was obtained from E. coli BL21 cells by the induction of IPTG, and the lipase production reached 192.2 U/mL in BL21[pET-PVL] after culture for 15 h at 15 ℃. The maximum production was obtained by culturing BL21 cells in LB medium (pH8.5) with 15 mg/mL glucose and 200 μg/mL ampicillin, as well as adding 100 μg/mL IPTG at OD600 of 1.2. A single protein band of 31 kDa was displayed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) after affinitive purification. The properties of lipase expressed in E. coli were similar to the native one，which could hydrolyze all three esters of triglyceride. [ Conclusion ] We have succeeded in over-expressing the lipase gene from P. vulgaris in E. coli, and this research has laid a foundation for improvement and industrial application of this lipase.