Method for building N-glycosylation efficiency detection receptor protein models in Escherichia coli by aid of skeleton proteins Fn3 (fibronectin type III domain)
A technology of Escherichia coli and skeleton protein, applied in microorganism-based methods, chemical instruments and methods, biochemical equipment and methods, etc., can solve problems such as inferior flexibility, improve physical and chemical properties, clarify structure-activity relationship, and achieve high efficiency and low cost. The effect of mass production
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Embodiment 1
[0018] 1. In this example, human fibronectin type III domain (Fn3) protein was used as a model gene (EMBL accession number AJ320527). At the 5′ end of the gene, 6 histidine residues are introduced, and at the FGloop region, a base sequence encoding the glycosylation site DQNAT is introduced. See the structure of the fusion gene. figure 1 . The gene was constructed into pIG6H by gene recombination, see the vector structure figure 2 .
[0019] 2. After the human fibronectin type III domain mutant Fn3-Gly-loop gene expression cassette sequence shown in the sequence table was synthesized (Nanjing GenScript Biotechnology Co., Ltd.), EcoRV and HindIII were used to construct an E. coli expression vector On pIG6H, the recombinant vector pIG6H-Fn3-Gly-loop was obtained.
[0020] 3. the expression vector constructed and pACYCpgl electric shock transformation CLM37 escherichia coli bacterial strain, then transformants were inoculated to the LB solid medium (per liter of culture) cont...
Embodiment 2
[0024] 1. In this example, human fibronectin type III domain (Fn3) protein was used as a model gene (EMBL accession number AJ320527). At the 5′ end of the gene, 6 histidine residues are introduced, and at the FGloop region, a base sequence encoding the glycosylation site DQNAT is introduced. See the structure of the fusion gene. figure 1 . The gene was constructed into pIG6H by gene recombination, see the vector structure figure 2 .
[0025] 2. After the human fibronectin type III domain mutant Fn3-Gly-loop gene expression cassette sequence shown in the sequence table was synthesized (Nanjing GenScript Biotechnology Co., Ltd.), EcoRV and HindIII were used to construct an E. coli expression vector On pIG6H, the recombinant vector pIG6H-Fn3-Gly-loop was obtained.
[0026] 3. the expression vector constructed and pACYCpgl electric shock transformation CLM37 escherichia coli bacterial strain, then transformants were inoculated to the LB solid medium (per liter of culture) cont...
Embodiment 3
[0030] 1. In this example, human fibronectin type III domain (Fn3) protein was used as a model gene (EMBL accession number AJ320527). At the 5′ end of the gene, 6 histidine residues are introduced, and at the FGloop region, a base sequence encoding the glycosylation site DQNAT is introduced. See the structure of the fusion gene. figure 1 . The gene was constructed into pIG6H by gene recombination, see the vector structure figure 2 .
[0031] 2. After the human fibronectin type III domain mutant Fn3-Gly-loop gene expression cassette sequence shown in the sequence table was synthesized (Nanjing GenScript Biotechnology Co., Ltd.), EcoRV and HindIII were used to construct an E. coli expression vector On pIG6H, the recombinant vector pIG6H-Fn3-Gly-loop was obtained.
[0032] 3. the expression vector constructed and pACYCpgl electric shock transformation CLM37 escherichia coli bacterial strain, then transformants were inoculated to the LB solid medium (per liter of culture) cont...
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