Influencing Factors On The Expression Level Of Recombinant Epitope-based Proteins In Escherichia Coli

Sine the middle of20century, the first recombinant protein, insulin, had beensuccessfully expressed in Escherichia coli, researchers explored the influencingfactors on the expression of recombinant protein. It had been reported that theinfluencing factors included host cell, expression vector, mRNA structure, codonsand fermentation condiction. Although some advanced expression platformsincluding prokaryotic and eukaryotic expression system provide the facilitation onthe expression of recombinant protein, a number of recombinant proteins are notsuccessfully expressed. In recent years, researchers have focused on the sequence ofamino acid and encoding gene which influence the expression of recombinantproteins in E.coli.In the reaserch of the activity of recombinant epitope-based proteins infoot-and-mouth disease virus (FMDV), we found that some recombinantepitope-based proteins were not expressed, and some were expressed in lower level.As we known, recombinant epitope-based protein is a non-nature protein that isreconstructed with different B cell epitopes and T cell epitopes with flexible aminoacid named linker. The amino acid composition of recombinant epitope-basedprotein is very specific because it is often on the surface of protein. Furthermore, thecodon bias between the encoding gene of FMDV and E.coli may be the one obstacleinfluencing the expression.In this study, to develop efficient recombinant vaccine against FMD, weanalyzed the amino acid sequences and nucleotide sequences of24FMDVmultiple-epitope-polypeptides (MEP) composed of multiple copies of B cell epitopesand Th cell epitopes of VP1protein of FMDV, and studied the relationship between expression level of recombinant FMDV epitope proteins (rFEPs) and thoseparameters like each amino acid composition, hydrophilic/hydrophobic amino acidcomposition, PI, GC/GC2content and CAI using Pearson correlation statisticanalysis. We found that high-level expression of the rEPs was attributed to:(1) a high content of Arg, Asn, Asp and Thr;(2) a low content of Gln, Pro and Lys;(3) a high content of hydrophilic amino acids and a higher isoelectric pointvalue resulting from abundant Arg;(4) the appropriate guanine and cytosine content (GC) in the encoding genes;(5) the GC2in the encoding genes.And we found that the high level expression of the rFEPs was not attributed to:(1) the content of hydrophobic amino acids, charge;(2) the GC1andGC3in the encoding genes;(3) the CAI and Nc in the encoding genes.Our statistical analyses reveal a number of surprising sequence correlations thathave evaded characterization via traditional mechanistic experimentation whichprovide a new angle to design epitope vaccine.