Refolding, Purification of the Recombinant Enerokinase and Construction of a Novel Expression Plasmid

Abstract: Enterokinase(EK) is a heterodimeric serine protease which plays a key role in initiating the proteolytic digestion cascade in the mammalian duodenum. The enzyme acts by converting trypsinogen into trypsin via a high specific cleavage following the pentapeptide recognition sequence (Asp)4-Lys. This stringent site specificity gives EK great potential as a fusion protein cleavage regent. Compared with proteases such as thrombin or factor Xa and chemicals such as CNBr or hydroxylamine, bovine enterokinase is an ideal choice because it can cleave with high specificity after the sequence (Asp)4-Lys, it can be tolerant to a wide range of conditions, and it allows any downstream fusion target protein to retain its native amino-terminus without leaving any unwanted amino acid residues on their amino termini.In this study, firstly, we continued to study the recombinant engineering bacteria pET39b-EKL/BL21(DE3) which have been structured successfully in our laboratory about the protein expression and purification. The suitable induction conditions were confirmed, the bacterium were cultivated under37℃, when the cell density(OD600) reached 0.7, IPTG was added, finally, the bacterium were induced with 0.5mmol/L IPTG for 6h. Protein DsbA-EK-(His)8 was expressed in prokaryotic cell successfully. After the affinity purification of the supernatant, the yield of the target protein- enterokinase was 43.65μg per 100 milliliter of fermentation liquid and the recovery rate was 8.34%. Because of the fusion protein was mainly expressed in the form of inclusion bodies and for the low recovery percents of EK in the supernatant, inclusion body was denatured before added to the column. After washing, the inclusion bodies were dissolved in a 8M urea solution(pH8.0) containing 100 mmol/L GSH and 50 mmol/L Tris-HCL. The dissolved protein was further purified and refolded by Ni2+ affinity column to obtain active enterokinase. Meanwhile, we still considered the different condition in the process of denaturation and dissolution to enhance the content of EK. The results indicated that the yield of the target protein- enterokinase after refolding and purification was 470μg per 100 milliliter of fermentation liquid and the recovery rate was 20.3%.In the second part, a new strategy about the expression of enterokinase was started, which is building a double-plasmid system. In this system, the target fusion protein can be digested without enterokinase additionally. One recombinant plasmid was builded for the fusion protein which exists in inclusion bodies. A suitable plasmid was choiced frist, which has a different replication orgin from the most commercial plasmid and a low copy number that will not affect the adequately expressing of target protein. The EK_LDNA fragment was insert into the plasmid pSB3K3. Subsequently, we expressed the recombinant plasmid pSB3K3-EK_Land studied the enzyme activity under influence of different conditions. The optimal reaction condition would be fixed: reaction buffer pH value was 7.5, reaction temperature was 35℃and reasonable reaction time was 2h…
Key words: Bovine Enterokinase Light Chain ; Fusion Protein; Affinity Purification ; Inclusion Body ; Double Plasmid System

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