Molecular Engineering Approaches For Improving Refolding Yield, Thermostability And Specificity Of Human Enteropeptidase

Enteropeptidase is a double chains serine protease which can hydrolyze the peptidebond at the C terminal of the sequence ‘DDDDK’. Due to the unique selectivity ofsubstrate, it is one of the important biotechnological tools. Up to now, enteropeptidasehas been widely used in the fields of fusion protein cleavage and protein analysis.Especially in the biopharmaceutical industry, enteropeptidase is needed for removingthe fusion tags in the down-stream process, because many recombinant protein drugsare expressed as fusion form. Compared to the full enzyme, the smaller light chain ofenteropeptidase containing the catalytic domain is more easily cloned and expressed.For the high available, the recombinant light chain of bovine enteropeptidase (bEPL) ismost commonly used. However, the recent studies showed that the activity of the lightchain of human enteropeptidase (hEPL) was10-fold higher than bEPL. Therefore,hEPL is thought to be a better biotechnological tool. However, the low refolding yieldhampers its application in large scale. On the other hand, similar to its analogs fromother mammumanian, hEPL may occasionally cleave the peptide bind at non-specificpositions and affect the following purification.According to the analysis of dihedral angles and detail position of every residues,and in combination with molecular dynamic simulations (MDS), several amino acidresidues were chosen to be replaced by proline residues. The software Amber9,Discovery Studio2.5.5and VMD played an important role in our study. Site-directmutagenesis was conducted to evaluate the proline’s effect on refolding yield. And thechange of thermostability was tested using comparison assays by residual activitiesanalysis after heating. On the other hand, the crystal structure of light chain of medakaenteropeptidase (mEPL) which has much higher specificity was solved by X-raydiffraction technology. And the critical residues for specificity were predicted by thestructural comparing among mEPL, bEPL and hEPL, and identified by site-directedmutations. Furthermore, new mutants of hEPL for higher specificity were designed andtested by active assays. After all, the mutations of K62P and K101P could obviously increase the refoldingyield of hEPL while the thermostability was also improved. On the other hand, thecrystal structure of mEPL was successfully solved. And according to the structuralalignment, two additional interactions (His24-Gly185and Glu136-Arg213) in mEPLwere considered as part reason for the high specificity. The interaction His24-Gly185was then introduced into hEPL(K62P/K101P), and improved the specificity asprediction. Nevertheless, the interaction Glu136-Arg213didn’t exhibit the same effectin hEPL. And it was demonstrated that the amino acid residue at the position136wasimportant for the activity of the enzyme.The present study targeted improvement of the thermostability, refolding yield andspecificity of hEPL. The bioengineered hEPL (K62P/K101P/L24H) can become usefultool for biotechnological application, especially in large-scale production of fusionproteins.