High-efficiency expression method of recombinant human endomucin in Escherichia coli
A technology of Escherichia coli and an expression method, applied in the field of biopharmaceuticals, can solve problems such as the high-efficiency expression of recombinant human Endomucin that has not yet been reported, and achieve the effect of high-efficiency expression
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Embodiment 1
[0025] 1) Clone the full-length human Endomucin gene into pGEX-2T expression plasmid. The restriction enzymes used are BamH I at the N-terminus and RcoRI at the C-terminus to connect the target gene to glutathione sulfhydryl transferase. After the (GST) gene, insert a thrombin cut site between the GST and Endomucin genes to construct a new cloning plasmid pGEX-Endomucin to express the GST-Endomucin fusion protein.
[0026] 2) Using Escherichia coli BL21(DE3)(plySs) strain as the host strain for expressing Endomucin protein, transforming the pGEX-Endomucin plasmid into E. coli BL21(DE3)(plySs) to construct an engineered E. coli strain capable of expressing GST-Endomucin fusion protein BL21-GEn.
[0027] 3) On the expression process of GST-Endomucin fusion protein, the specific process steps are:
[0028] ① Streak the E. coli engineering strain BL21-GEn to inoculate the LB semi-solid medium (containing 100ug / mL Amp), invert the culture overnight at 37°C, until a single colony of the e...
Embodiment 2
[0035] 1) Clone the full-length human Endomucin gene into the pGEX-2T expression plasmid. The restriction enzymes used are BamH I at the N-terminus and RcoRI at the C-terminus to connect the target gene to glutathione sulfhydryl transferase. After the (GST) gene, insert a thrombin cleavage site between the GST and Endomucin genes to construct a new cloning plasmid pGEX-Endomucin to express the GST-Endomucin fusion protein.
[0036] 2) Using Escherichia coli BL21(DE3)(plySs) strain as the host strain for expressing Endomucin protein, transforming the pGEX-Endomucin plasmid into E. coli BL21(DE3)(plySs) to construct an engineered E. coli strain capable of expressing GST-Endomucin BL21-GEn.
[0037] 3) In the expression process of GST-Endomucin fusion protein, the specific process steps are:
[0038] ① Streak the E. coli engineering strain BL21-GEn to inoculate the LB semi-solid medium (containing 100ug / mL Amp), invert the culture overnight at 37°C, until a single colony of the enginee...
Embodiment 3
[0045] 1) Clone the full-length human Endomucin gene into the pGEX-2T expression plasmid. The restriction enzymes used are BamH I at the N-terminus and RcoRI at the C-terminus to connect the target gene to glutathione sulfhydryl transferase. After the (GST) gene, insert a thrombin cleavage site between the GST and Endomucin genes to construct a new cloning plasmid pGEX-Endomucin to express the GST-Endomucin fusion protein.
[0046] 2) Using Escherichia coli BL21(DE3)(plySs) strain as the host strain for expressing Endomucin protein, transforming the pGEX-Endomucin plasmid into E. coli BL21(DE3)(plySs) to construct an engineered E. coli strain capable of expressing GST-Endomucin BL21-GEn.
[0047] 3) In the expression process of GST-Endomucin fusion protein, the specific process steps are:
[0048] ① Streak the E. coli engineering strain BL21-GEn to inoculate the LB semi-solid medium (containing 100ug / mL Amp), invert the culture overnight at 37°C, until a single colony of the enginee...
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