融合超嗜热菌Pyrococcus furiosus红素氧还蛋白可提高靶蛋白的热稳定性

doi:10.13560/j.cnki.biotech.bull.1985.2021-0060

摘要/Abstract

摘要：

热稳定性是功能蛋白（特别是酶蛋白）储存和使用的重要限制因子。许多商业酶因耐热性不能达到工业生产的热处理条件而无法被采用;另外,植物中与光合作用有关的关键酶也受高温抑制,从而导致农作物产量下降。因此,对酶蛋白进行分子改良以提高其热稳定性,进而提升其功效和扩大其应用范围,具有重要的现实意义。选取3种典型的热稳定性差、易聚集但具有重要功能的酶蛋白（小桐子抗坏血酸过氧化物酶1即JcAPX1、大肠杆菌高丝氨酸-O-转琥珀酰酶即EcMetA、假单胞菌亚磷酸盐脱氢酶即PsPtxD）作为靶蛋白,鉴定出超嗜热菌Pyrococcus furiosus的小分子红素氧还蛋白（rubredoxin）能够作为热稳定融合标签,提高大肠杆菌重组表达靶蛋白的可溶性、热稳定性及其活性的耐热能力,为通过融合表达策略提高目标蛋白的热稳定性并强化其应用提供了一个新的技术选择。

关键词: 蛋白热稳定性, 融合标签, 超嗜热菌, Pyrococcus furiosus, 红素氧还蛋白

Abstract:

Thermostability is an important restriction factor for the storage and use of functional proteins（especially enzymatic proteins）. Many commercial enzymes are not suitable for use because their heat tolerance usually cannot meet the heat processing conditions of industrial production. In addition,some key enzymes involved in plant photosynthesis are also inhibited by high temperature,thus leading to the decrease of crop yield. Therefore,it is of great practical significance to improve the thermostability of enzyme proteins by molecular modification so as to enhance their efficacy and expand their application. Herein,we chose three important enzymes that are canonically heat-labile or prone to aggregation,including JcAPX1（cytosolic ascorbate peroxidase 1 of Jatropha curcas）,EcMetA（homoserine-O-transsuccinidase of Escherichia coli）,and PsPtxD（phosphite dehydrogenase of Pseudomonas sp.）,and used them as target proteins and identified the small rubredoxin from hyperthermophile Pyrococcus furiosus as thermo-stable fusion tag. It could be used to improve the solubility,thermostability and active heat-tolerance of those recombinant target proteins expressed in E. coli. Prospectively,this might provide a new technique choice for improving the thermostability and related applications of target proteins through the fusion expression strategy.

Key words: protein thermostability, fusion tag, hyperthermophile, Pyrococcus furiosus, rubredoxin

吴娇, 余桂珍, 袁航, 刘娴, 高艳秀, 龚明, 邹竹荣. 融合超嗜热菌Pyrococcus furiosus红素氧还蛋白可提高靶蛋白的热稳定性[J]. 生物技术通报, 2021, 37(10): 110-119.

WU Jiao, YU Gui-zhen, YUAN Hang, LIU Xian, GAO Yan-xiu, GONG Ming, ZOU Zhu-rong. Improvement on the Thermostability of Target Proteins by Fusing Rubredoxin from Hyperthermophile Pyrococcus furiosus[J]. Biotechnology Bulletin, 2021, 37(10): 110-119.

图/表 7

表1 本研究中所用引物

Table 1 Primers used in this study

名称Name	序列Sequence（5'-3'）	备注Remarks
Rub-F2	TCTATGATGAAGACGCTGGGATCCTGACAATGGTATTTCACCTGGAACTAAGTTTGAG
Rub-R2	GCACCACAAATAGGACAGACCCAATCATCTGGTAGCTCCTCAAACTTAGTTCCAGGTG
Rub-F1Sa	GGCAGTCGACGCTAAGTGGGTTTGTAAGATATGCGGATACATCTATGATGAAGACGCTG	Sal I
Rub-R1Xh	GCGACTCGAGATCTTCTAACTTTTCAAATTCACTCTTAGGAGCACCACAAATAGGACAG	Xho I
JcAPX1-5Nd	GCGGTCATATGGCTAAGAACTATCCAAAAG	Nde I
PsPtxD-5Nd	GCTATCATATGGCTCTGCCGAAACTCGT	Nde I
EcMetA-5Nd	GCGTTCATATGCCGATTCGTGTGCC	Nde I

图1 Rub基因的PCR合成及其融合表达载体的构建 A:Rub基因PCR;B、C、D:重组克隆pET（JcAPX1-Rub）、pET（PsPtxD-Rub）和pET（EcMetA-Rub）的菌落PCR鉴定; M:DNA marker;箭头标示目标PCR条带

Fig.1 PCR synthesis of the Rub gene and construction of its fusion expression vectors A:Rub gene PCR;B,C,D:Colony PCR identification of vector pET（JcAPX1-Rub）,pET（PsPtxD-Rub）,and pET（EcMetA-Rub）,respectively. M:DNA marker. Arrows indicate the target PCR bands

表2 Rub、靶蛋白及其融合蛋白的特性

Table 2 Properties of Rub,target proteins,and their fusion forms

名称 Name	氨基酸数目 Number of amino acids	分子量 Molecular weight/kD	等电点 pI	pH 7.0条件下的净电荷 Net charge at pH 7.0
Rub	53（+LE）	6.14	4.02	-9
JcAPX1	258	28.56	5.76	-10
JcAPX1-Rub	313	34.7	5.13	-19
EcMetA	317	36.79	5.36	-17
EcMetA-Rub	372	42.9	4.92	-26
PsPtxD	345	37.55	5.67	-11
PsPtxD-Rub	400	43.65	5.06	-20

图2 融合Rub可促进靶蛋白JcAPX1、PsPtxD、EcMetA在大肠杆菌中的可溶性表达通过SDS-PAGE分析（A、D、G）、基于蛋白条带灰度的可溶性估测（B、E、H）以及菌裂解液的颜色显示（C、F、I）,分别成对比较重组靶蛋白及其Rub融合蛋白的可溶性表达,即 A-C:JcAPX1相比JcAPX1-Rub（37℃诱导4 h）;D-F:PsPtxD相比PsPtxD-Rub（25℃诱导过夜）;G-I:EcMetA相比EcMetA-Rub（30℃诱导8 h）。箭头标示重组蛋白;M:蛋白质分子量标准;UI:未诱导细菌;T:诱导细菌裂解总蛋白;P:“T”的沉淀;S:“T”的上清

Fig. 2 Rub fusion enhances the soluble expressions of target proteins JcAPX1,PsPtxD,and EcMetA in E. coli SDS-PAGE analysis（A,D,and G）,protein band densitometry-based solubility estimation（B,E,and H）,and color visualization of bacterial cell lysate（C,F,and I）were used to pairwise compare the soluble expressions of recombinant target proteins and their Rub-fusion forms,i.e. A-C:JcAPX1 vs. JcAPX1-Rub（37℃/4 h induction）;D-F:PsPtxD vs. PsPtxD-Rub（25℃/overnight induction）;G-I:EcMetA vs. EcMetA-Rub（30℃/8 h induction）. Arrows indicate the recombinant proteins. M:Protein molecular weight marker. UI:Cell lysate of the uninduced bacteria. T:Cell lysate of the induced bacteria. P:The pellet fraction of “T”. S:The soluble fraction of “T”

图3 融合Rub可增强靶蛋白JcAPX1、PsPtxD、EcMetA的热稳定性通过SDS-PAGE分析（A、C、E）和基于蛋白条带灰度的可溶性估测（B、D、F）,分别成对比较重组靶蛋白及其Rub融合蛋白热处理后的可溶性变化（热稳定性）,即 A、B:JcAPX1相比JcAPX1-Rub;C、D:PsPtxD相比PsPtxD-Rub;E、F:EcMetA相比EcMetA-Rub。箭头标示重组蛋白;M:蛋白质分子量标准;CK:未处理样品;S、P:每个热处理样品离心后的上清和沉淀

Fig. 3 Rub fusion improves the thermostability of target proteins JcAPX1,PsPtxD,and EcMetA SDS-PAGE analysis（A,C,and E）and protein band densitometry-based solubility estimation（B,D,and F）were used to pairwise compare the heat-induced solubility changes（i.e. thermostability）of recombinant target proteins and their Rub-fusion forms,i.e. A,B:JcAPX1 vs. JcAPX1-Rub;C,D:PsPtxD vs. PsPtxD-Rub;E,F:EcMetA vs. EcMetA-Rub. Arrows indicate the recombinant proteins. M:Protein molecular weight marker. CK:Untreated sample. S,P:The soluble,pellet fraction of each heat-treated sample,respectively

图4 融合Rub可提高JcAPX1、PsPtxD体外酶活的耐热性通过胶活性染色（A）和分光光度法测定（B）,比较JcAPX1及其Rub融合形式JcAPX1-Rub热处理后的酶活性变化;同样,用分光光度法分析比较PsPtxD及其 Rub融合形式PsPtxD-Rub热处理后的酶活性变化（C）。CK:未处理样品;S、P:每个热处理样品离心后的上清和沉淀

Fig. 4 Rub fusion improves the heat tolerance of the in vitro enzymatic activities of JcAPX1 and PsPtxD The heat-induced enzymatic activity changes of JcAPX1 and its Rub fusion JcAPX1-Rub were compared by the analyses using in gel activity staining（A）and spectrometric assay（B）. Likewise,the spectrometric assay was also used for the suite of PsPtxD and PsPtxD-Rub（C）. CK:Untreated sample;S,P:the soluble,pellet fraction of each heat-treated sample,respectively

图5 细菌稀释点板分析Rub融合对EcMetA重组大肠杆菌在高温胁迫下的生长影响

Fig. 5 Influence of Rub fusion on the growth of EcMetA recombinant E. coli strain under heat stress by bacterial dot plating test with serial dilution

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