Effect of Gene Co-expression on Heterologous Soluble Expression，Purification and Enzymatic Properties of Human LysoPLD
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摘要: 目的：为实现人源溶血磷脂酶D（LysoPLD）的原核异源可溶性表达。方法：通过NCBI检索，确定人源LysoPLD基因序列（GenBank: L46720.1）。采用密码子优化后的序列，克隆至pET-28a表达载体中，采用共表达麦芽糖结合蛋白融合标签（MBP）和共表达促蛋白正确折叠分子伴侣触发因子Trigger factor（tig）两种方式提高LysoPLD蛋白在大肠杆菌中的异源可溶性表达，建立对应蛋白的纯化工艺包括离子柱纯化，硫酸铵盐析，疏水柱纯化，淀粉树脂柱（Amylose Resin）纯化，分离纯化获得的重组酶，经聚丙烯酰胺凝胶电泳（SDS-PAGE）测定蛋白纯度，以对羟基棕榈酸酯为底物对比两种蛋白的酶学性质。结果：成功构建载体pET28a-MBP-LysoPLD和pET28a-pTF16-LysoPLD，并获得工程菌BL21（DE3）-pET28a-MBP-LysoPLD和BL21（DE3）-pET28a-pTf16-LysoPLD。BL21（DE3）-pET28a-MBP-LysoPLD经0.6 mmol/L异丙基硫代半乳糖苷（IPTG）低温诱导过夜可获得上清表达的MBP-LysoPLD蛋白；BL21（DE3）-pET28a-pTf16-LysoPLD在含有0.5 μg/mL L-Arabinose的LB培养基中培养，经0.1 mmol/L IPTG低温诱导表达，可获得可溶性表达的LysoPLD蛋白，经纯化，酶纯度可大于80%。以对羟基棕榈酸酯为底物，对比两种方法得到的蛋白的酶学性质，发现二者催化反应的最适温度、最适pH、最适Ca2+浓度、比酶活基本一致。结论：两种基因共表达方式都可实现人源LysoPLD的在大肠杆菌中的可溶性表达，且酶学性质基本相同。
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关键词:
- 人源溶血磷脂酶D /
- 异源可溶性表达 /
- 麦芽糖结合蛋白融合标签 /
- 触发因子 /
- 酶学性质
Abstract: Objective:This study aimed to express soluble human LysoPLD in Escherichia coli.Methods:Human LysoPLD gene sequence (GenBank: L46720.1) was retrieved from NCBI and synthesized chemically. The codon optimized sequence was cloned into pET-28a vector and transformed to the E.coli BL2(DE3) strain which was co-expressed with maltose binding protein fusion tag (MBP) or co-expressed with a pTF16 chaperone plasmid, then induced by isopropyl thiogalactoside (IPTG). The downstream purification process was established, including ion column elution, ammonium sulfate salting out, hydrophobic column purification, and amylose resin purification. The LysoPLD purity was determined by polyacrylamide gel electrophoresis (SDS-PAGE) and the enzymatic properties were determined by catalytic reaction of p-hydroxypalmitate.Results:The plasmids pET28a-MBP-LysoPLD and pET28a-pTf16-LysoPLD were constructed, then transformed into the E.coli BL21(DE3) strain respectively. BL21(DE3)-pET28a-MBP-LysoPLD was induced by 0.6 mmol/L IPTG, and incubated overnight to obtain MBP-LysoPLD protein; BL21(DE3)-pET28a-pTF16-LysoPLD was cultured in LB medium containing 0.5 µg/mL L-arabinose then induced by 0.1 mmol/L IPTG, the soluble LysoPLD was obtained. After downstream purification, the purity of the human LysoPLDs expressed through both methods were more than 80%. It was found that using p-hydroxypalmitate as substrate, the optimum reaction conditions including optimal temperature, pH and Ca2+ concentration for both LysoPLDs were basically the same.Conclusion:In this study, two different methods for improving soluble expression of human LysoPLD in E.coli were established and proved to be successful. The purified human LysoPLDs were enzymatically active, and the enzymatic activity were basically the same. -
图 1 MBP基因、pET28a-LysoPLD载体骨架和pET28a-MBP-LysoPLD基因的PCR扩增电泳图
注：M：DNA Marker；泳道1（或2）：PCR扩增的电泳条带;A：MBP基因的PCR扩增片段；B：pET28a-LysoPLD载体骨架的PCR扩增片段；C：pET28a-MBP-LysoPLD中PCR扩增片段鉴定电泳图。
Figure 1. PCR amplification and identification of MBP gene、pET28a-LysoPLD and pET28a-MBP-LysoPLD
图 2 大肠杆菌表达的重组PLD和MBP-LysoPLD的SDS-PAGE分析
注：A中泳道1～4分别代表大肠杆菌BL21（DE3）诱导表达前全菌，诱导表达后全菌，诱导表达后超声破碎离心得到的上清，诱导表达后超声破碎离心得到的沉淀；B中泳道1～4分别代表分别代表大肠杆菌BL21（DE3）诱导表达前全菌，诱导表达后全菌，诱导表达后超声破碎离心得到的沉淀，诱导表达后超声破碎离心得到的上清。
Figure 2. Analysis of the recombinant of LysoPLD and MBP-LysoPLD expressed in E. coli by SDS-PAGE
图 3 大肠杆菌表达的重组LysoPLD（tig）的SDS-PAGE分析
注：M：Marker；1：诱导表达前全菌；2：诱导表达后全菌；3：诱导表达后超声破碎离心后得到的上清；4：诱导表达后超声破碎离心后得到的沉淀。
Figure 3. Analysis of the recombinant of pTf16-LysoPLD（tig）expressed in E.coli by SDS-PAGE
图 4 Amylose Resin纯化洗脱MBP-LysoPLD
注：M:Marker;1：上Amylose亲和柱前总蛋白；2：经Amylose亲和柱流穿的蛋白；3：经Amylose-B洗脱收集的蛋白。
Figure 4. Purification and elution of MBP-LysoPLD by Amylose Resin
图 5 蛋白纯化后的SDS-PAGE电泳图
注：A：SP琼脂糖凝胶柱LysoPLD蛋白洗脱SDS-PAGE电泳图；泳道1～5分别为1：Marker；2：上SP琼脂糖凝胶柱前总蛋白；3：经SP琼脂糖凝胶柱流穿的蛋白；4：经SP-B洗脱后收集的蛋白；5：经SP-B洗脱后收集的蛋白;（B）：阴离子柱（Q柱）LysoPLD蛋白洗脱SDS-PAGE图；1，Marker；2，经Q-B洗脱后收集的蛋白；3，Q-B洗脱后收集的蛋白。
Figure 5. SDS-PAGE analysis of purified PLD protein
图 6 LysoPLD（tig）与MBP-LysoPLD在不同温度相对酶活（A）和LysoPLD（tig）在不同金属离子下相对酶活（B）
Figure 6. Relative enzyme activities of LysoPLD (tig) and MBP-LysoPLD at different temperature (A) and LysoPLD (tig) at different metal ions (B)
图 7 PLD（tig）与MBP-LysoPLD在不同pH和浓度Ca2+下相对酶活
Figure 7. Relative enzyme activities of LysoPLD (tig) and MBP-LysoPLD at different pH and Ca2+ concentration
表 1 两种LysoPLD酶活性质比较
Table 1. Comparison of two LysoPLD enzyme activity
指标 MBP-PLD LysoPLD（tig） 总蛋白量（mg） 4.91 ± 0.03 5.79 ± 0.11 总活力（U） 20.98 ± 2.34 42.61 ± 3.05 比酶活（U/mg） 4.27 ± 0.02 7.32 ± 1.24 
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