Effects Of Hyper-acidic Protein Fusion Partners On The Soluble Expression Of Recombinant Proteins In Escherichia Coli

Protein engineering has been an important aspect in biotechnological research.In scientific area,to gain large amount of pure,soluble and bioactive protein is the prerequisite for studying its physiochemical properties and function.However, obtainment of natural protein cannot meet actual requirements,due to a few of strict limitations such as rare resources and high cost.With the development of protein engineering and genetic engineering,the recombinant proteins,being increasingly closer to their natural forms in terms of the physical,chemical and biological features, can be prepared in large scales.Although mammalian cells have been frequently applied to produce recombinant proteins since the 1990s,the Escherichia coli(E.coli) expression system is the most commonly used so far.This system is of many advantages including well-known genetic background,simple culture conditions, high-level expression,inexpensiveness and convenience.However,the E.coli expression system does have limitations.When a target protein is complicated and is of eukaryotic sources,this system often fails in making the expressed protein fold correctly,leading to formation of insoluble and non-active aggregates termed inclusion bodies.Functional recovery of proteins from their inclusion bodies usually should undergo a complex and inefficient process of denaturalization and refolding. Therefore,to maximize the soluble expression of recombinant protein in E.coli is certainly of high academic value and wide range of applications.Regarding this,some effectual approaches have been explored at present,in which the fusion expression strategy by using protein fusion partners(or tags)has been prevalently used.However,such fusion tags are not effective for all proteins.In this work,we were committed to finding common fusion partners to improve the soluble expression of target proteins(especially those recalcitrant to conventional solubilizing efforts)in E.coli.To this purpose,three candidate proteins with the features of E.coli origin,relatively small size and extremely high acidity were selected,including Msb(an E.coli acidic protein MsyB),Yd(one hypothetical protein,yjgD),and Od(the N-terminal domain ofσ70 factor of RNA polymerase, rpoD).They were tested as fusion tags for three highly aggregation-prone target proteins including EK(the bovine enterokinase),TEV(the tobacco etch virus protease) and Rb(rbcL,the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase). Meanwhile,other frequently used solubility-enhancing fusion tags such as Trx(TrxA), MBP,NusA and SUMO,were included for comparison.By using PCR amplification and restriction enzyme digestion,DNA fragments of fusion tags Msb,Yd,Od,MBP,NusA,Trx and SUMO were obtained and respectively fused with the genes of target proteins to generate series of expression vectors on the basis of backbone plasmid pET-32a(+).All of these vectors were expressed in E.coli BL21(DE3)by induction and subsequently analyzed by SDS-PAGE.The results demonstrated that all three hyper-acidic protein fusion tags Yd,Od as well as Msb could remarkably enhance the soluble expression of three highly aggregation-prone target proteins Ek,TEV and Rb.In addition,hybrid fusion tags such as MS,YS,OS, PS and NS(consisting of Msb,Yd,Od,MBP,and NusA,and the common His₆-tagged SUMO,respectively)were created,the acidic ones of which were also found to effectuate apparently in solubility enhancement for target proteins.Taken together,all these experimental results are well in consistence with the theoretical solubility prediction by the revised algorithm of Wilkinson-Harrison.We further evaluated the functional roles of all three hyper-acidic fusion partners (Yd,Od and Msb)by taking advantages of the yeast SUMO/Ulpl reaction and TEV auto-cleavage.Our results indicated that these hyper-acidic fusion partners actually act as intramolecular chaperones assisting in the correct folding of the target proteins, during their solubilising process.Conclusively,the hyper-acidic proteins(Msb,Yd,Od)in our study could be used as a category of universal and potent fusion tags to improve the soluble expression of target proteins in E.coli,and largely believed to confer broad prospects in the fields of proteins studies and biotechnological applications.