Construct Recombinant Prokaryotic Expression Vector For Antarctic Krill Trypsin Gene And Efficiently Expressed Trypsin In Escherichia Coli

Antarctic krill, also known as Euphausia superba, belongs to Arthropoda CrustaceaEuphausia. This shrimp-like invertebrates are the key species of the Antarctic region, mainlydistribute around the polar and concentrate in the Antarctic Ocean.They are one of the mostsuccessful animal species that have the maximum biomass energy and adapt to theenvironment.The extreme cold environment created a lot of active substances in the body of Antarctickrill. All these active substances have high research value and utilization prospects.The mostprominent of these features is the cold adapted trypsin-like activity enzyme. Antarctic krilltrypsin has been widely applied in many medical fields such as clearing body necrotic tissue,treating corneal alkali burn and removing off plaque. The Antarctic krill trypsin has graduallybecame a hotspot of the subject of light industry, nutrition and pharmacy.Due to the practical difficulties and high costs of obtainning krill samples, we usegenetic engineering techniques to express the Antarctic krill trypsin gene in E.coli. Our workis helpful to further study the properties of Antarctic krill trypsin.The Genomic DNA was extracted from the frozen krill obtained from Antarctic expedition,The trypsin gene was cloned using TAIL-PCR technology. Based on the conserved genesequences, gene specific primers were designed to transcribe mRNA into cDNA. The clonedtrypsin gene was constructed into the pGEX-2T prokaryotic expression vector, and thentransformed into E.coli BL21. The culture conditions were optimized in order to highlyexpress trypsin gene. The purification of trypsin from E.coli BL21was also optimized. 【Methods】(1) Take Antarctic krill mRNA as template, using the degenerate primers and RT-PCRtechnology to obtain the cDNA sequence of Antarctic krill trypsin gene (TRY).(2) Contruct recombinant prokaryotic expression vector pGEX-2T-TRY, and transfer therecombinant vector into the E.coli BL21, the recombinant pGEX-2T-TRY vector wasidentified by PCR and restriction enzyme digestion.(3) The identified recombinant pGEX-2T-TRY vector was transformed into E.coli BL21. TheTRY gene was highly expressed after optimizing the IPTG induced conditions.(4) The trypsin was pufired from E.coli BL21which highly expressed TRY using GlutathioneSepharose4B. The enzyme activity of purified trypsin was measured using BApNA by themethod of Bradford.【Results】(1) Successfully cloned the cDNA of Antarctic krill trypsin gene by TAIL-PCR. DesinedTRY gene specific primers from cloned cDNA sequence and successfully transcribedmRNA into cDNA.(2) Successfully constructed the recombinant prokaryotic expression vector pGEX-2T-TRY(3) E.coli BL21expressed TRY gene was inducted by IPTG. The SDS-PAGE electrophoresisresults revealed that the fusion GST-trypsin protein was successfully expressed. Themolecular weight of the fusion GST-trypsin protein on polyacrylamide gel is about55KDa, consisting with the expected results.(4) Glutathione Sepharose4B was used to purify the fusion GST-trypsin protein. But theenzyme activity was not detected.【Conclusion】We sucessfully construct the recombinant prokaryotic expression vector pGEX-2T-TRY.The Antarctic krill trypsin fused with GST tag was highly expressed in E.coli BL21. Wesuccessfully purified the fusion GST-trypsin protein and measured the enzyme activity,however, the enzyme activity was not detected.