Improvement on the Thermostability of Target Proteins by Fusing Rubredoxin from Hyperthermophile Pyrococcus furiosus

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

Abstract

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

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.

Figures/Tables 7

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

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

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

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”

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

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

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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