Clone And Expression Of Bovine Recombinant Enterokinase Catalytic Subunit In Escherichia Coli

Enterokinase is a kind of heterodimer serine proteinase which reside in mammal introduodenal. Enterokinase is protrypsin physiology activating agent. In vivoenterokinase make effort in activation from protrypsin to trypase,which is an importantactivating agen of many digestive system enzyme precursor. So enterokinase isregarded as an important initiation enzyme of digestive system.Enterokinase offen seem to reside in intestinal brush border cellular membrane. Patho research acquire that duodenum and pancreatic recirculation containing a great quantity of enterokinase will activiate protrypsinresulting into acute pancreatitis. Body lack EK could take on diarrhea ,vomit andswollen,also result in dysplasia ,blood protein insufficient and anaemia.Enterokinase molecular weight is kDa.It is composed of a 115kDa H-chain and a 35kDa L-chain. Enterokinase specificall hydrolyze protein substrate in pH4.5～9.5 and4～45℃.The polypeptide from EK spilt fusion protein has the same amino acid sequence of N-terminal with the wild type.With this feature EK is regarded as an important tool in fusion protein express modification.Natural EK is composed of a structure subunit and a catalysis subunit which combine them With a intermolecular disulfide bond.The structure subunit fixes the catalysis subunit at intestinal brush border and lead it to enteric cavity.The catalysis subunit could specially recognize Asp-Asp-Asp-Asp-Lys and slice it at carboxyl terminus.Then it active protrypsin to trypsin and start enzyme precursor activation.EK L-chain sequence keep conservation Homo sapien,bovine and pig.It's recognition sequence also keeps conservation in vertebrate.Also all protrypsin has feature of four asparaginate bind,which seems rare in other natural protein.EK's active center has a special basicion site and it could bond with Asp-Asp-Asp-Asp.EK L-chain has holoenzyme slice activation in extraorgan experiment.Natural EK is difficult to obtain,it's source is limited.At present it is offen obtained from separation and extraction in animal's duodenum.This method is difficult to operate and cost too much,also EK obtained with this method is apt to be polluted by other enzymes.With respect to above reasons,we adopt E coli expression system to colone and express EKL-chain gene.To easy to separate purpose protein,we construct GST-EKL fusion expression vector to high- efficiently express EK.In order to obtain the gene of bovine enterokinase light chain ,which would be used in the cleavageand purification of fusion proteins. The fragment of bovine enterokinase light chain cDNA was obtained by RT-PCR from bovine′s duodenal mucosa , then cloned into the pMD18-T cloning vector and sequenced. Compared with the sequence deposited in GenBank ,the cloned gene sequence is correct. Then the interested gene fragment was inserted into the pGEX-2T expression plasmid. The recombinant vector pGEX-rEKL was transformed into E.coli BL21 and induced by IPTG. It was confirmed that the nucleotide sequence was correct on the conjunction site between the recombinant DNA 5′terminal multi2cloning site and recombinant fragment after the analysis of the nucleotide sequence. SDS-PAGE analysis indicated that target product was about 61 kDa.The research succussfully clone and express EKL-GST fusion protein in Ecoli This investigation would be able to lay a foundation for enterokinase activity research and farther application of expression products on a large scale.