基因共表达对人源LysoPLD异源可溶性表达、纯化及酶学性质的影响

马文君 滕琳 王培培 卫宏远 郑春阳

马文君,滕琳,王培培,等. 基因共表达对人源LysoPLD异源可溶性表达、纯化及酶学性质的影响[J]. 食品工业科技,2021,42(7):102−109. doi:  10.13386/j.issn1002-0306.2020060091
引用本文: 马文君,滕琳,王培培,等. 基因共表达对人源LysoPLD异源可溶性表达、纯化及酶学性质的影响[J]. 食品工业科技,2021,42(7):102−109. doi:  10.13386/j.issn1002-0306.2020060091
MA Wenjun, TENG Lin, WANG Peipei, et al. Effect of Gene Co-expression on Heterologous Soluble Expression,Purification and Enzymatic Properties of Human LysoPLD[J]. Science and Technology of Food Industry, 2021, 42(7): 102−109. (in Chinese with English abstract). doi:  10.13386/j.issn1002-0306.2020060091
Citation: MA Wenjun, TENG Lin, WANG Peipei, et al. Effect of Gene Co-expression on Heterologous Soluble Expression,Purification and Enzymatic Properties of Human LysoPLD[J]. Science and Technology of Food Industry, 2021, 42(7): 102−109. (in Chinese with English abstract). doi:  10.13386/j.issn1002-0306.2020060091

基因共表达对人源LysoPLD异源可溶性表达、纯化及酶学性质的影响

doi: 10.13386/j.issn1002-0306.2020060091
详细信息
    作者简介:

    马文君(1990−),女,博士研究生,研究方向:食品微生物,E-mail:mwj9061@163.com

  • 中图分类号: Q 789

Effect of Gene Co-expression on Heterologous Soluble Expression,Purification and Enzymatic Properties of Human LysoPLD

  • 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。
  • 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-PLDLysoPLD(tig)
    总蛋白量(mg)4.91 ± 0.035.79 ± 0.11
    总活力(U)20.98 ± 2.3442.61 ± 3.05
    比酶活(U/mg)4.27 ± 0.027.32 ± 1.24
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  • 收稿日期:  2020-06-09
  • 网络出版日期:  2021-01-28
  • 刊出日期:  2021-04-01

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