Improvement On The Thermostability Of Target Proteins By Fusing Rubredoxin From Hyperthermophile Pyrococcus Furiosus

Thermostability is an important restriction factor for the storage and use of functional proteins,especially for enzymatic proteins.Many commercial enzymes are not suitable for use because their heat tolerance can not meet the heat processing conditions of industrial production(usually over 45 oC).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 enzymatic proteins by molecular modification so as to enhance their efficacy and expand their application.At present,there are two main categories of molecular approaches for improving protein thermostability: one is chemical engineering strategy(i.e.chemical modification),and the other is genetic engineering strategy(i.e.DNA mutagenesis or protein fusion strategy).Among them,protein fusion strategy has recently emerged as a potent manner to improve the solubility and thermostability of target proteins,which mainly depends on the availability of effectual fusion tags or adaptors.Therefore,herein we chose three important enzymes that are distinctly heat-labile or prone to aggregation,including JcAPX1(cytosolic ascorbate peroxidase 1 of Jatropha curcas),EcMetA(homoserine-O-transsuccinidase of Escherichia coli),and PsPtx(phosphite dehydrogenase of Pseudomonas sp.),and used them as target proteins to evaluate the small rubredoxin from hyperthermophile Pyrococcus furiosus(termed PfRub).We hoped to verify whether or not PfRub could be used as a thermostabilizing fusion adaptor to improve the solubility,thermostability and activity heat-tolerance of these target proteins recombinantly expressed in E.coli.The main results are as follows:(1)Gene synthesis of fusion adaptor PfRub and construction of its prokaryotic fusion expression vectorsFirstly,the DNA fragment of PfRub was directly synthesized by primer complementary extension,then double digested by Sal I and Xho I after purification,and subsequently inserted into the Xho I site behind the 3'-terminus of the coding genes of target proteins in vectors pET(JcAPX1),pET(EcMetA)and pET(PsPtx),respectively.Three prokaryotic fusion expression vectors were thus constructed and further verified by sequencing.(2)Fusing with PfRub can improve the solubility,thermostability and activity heat-tolerance of JcAPX1The results from SDS-PAGE analyses showed that the solubility of recombinant JcAPX1 could be increased from 12% to 94%,and its thermostability could be remarkably enhanced by C-terminal fusion with PfRub.Meanwhile,the results of APX gel activity staining and spectrometric assay demonstrated that this PfRub fusion could also improve the heat tolerance of JcAPX1 enzymatic activity to a degree of at least 3 oC increase.(3)Fusing with PfRub can improve the solubility and thermostability of EcMetA as well as the heat tolerance of its recombinant E.coli strainSimilarly,C-terminal fusion of PfRub could effectively improve the solubility(with nearly 20% increase)and thermostability of recombinant EcMetA.In addition,bacterial dot plating test with gradient dilutions showed that the colony growth of EcMetA-PfRub recombinant E.coli strain on M9 basic medium was significantly better than that of EcMetA alone after 2 h of heat treatment at 42 oC,48 oC,and 52 oC,implying that this PfRub fusion could also effectively improve the heat tolerance of EcMetA recombinant E.coli cells.(4)Fusing with PfRub can improve the solubility,thermostability and activity heat-tolerance of PsPtxAs seen from SDS-PAGE analyses,the solubility and thermostability of recombinant PsPtx protein were significantly enhanced by fusing PfRub at C-terminal.Meanwhile,the results of gel activity staining showed that PfRub fusion could also effectively protect PsPtx from thermal inactivation,and this was confirmed by a more sensitive spectrometric enzymatic assay that the heat tolerance of PsPtx activity could be increased by at least 12 oC.In conclusion,PfRub,as a fusion adaptor,not only improves the solubility and thermostability of target proteins,but also protects them from thermal inactivationand confers a better heat tolerance in its recombinant bacterial strains.PfRub is derived from hyperthermophile Pyrococcus furiosus,and its thermostabilizing role might be attributed to its ultra-high thermostability and intramolecular cis-transfer to fused target proteins.Therefore,this work seems to provide a new and effective route to enhance the thermostability and expand the applications of target proteins by using fusion strategy.In particular,this approach might also be suitable for plant transgenic studies with a high feasibility to improve the heat stress tolerance of plants.