Leader sequence for higher expression of recombinant proteins
A technology of sequence and leader peptide, applied in the field of novel leader sequence
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Embodiment 1
[0136] Example 1: Construction of plasmid pET28aULL1INS
[0137] The gene encoding proinsulin along with the nucleotide sequence of SEQ ID NO: 9 encoding the peptide ULL1INS was designed, codon optimized and chemically synthesized, and cloned in pUC57 to make pUC57ULL1INS. The gene fragment was cloned into pET28a vector. Restriction digestion of the pUC57ULL1INS plasmid was performed by setting up a reaction mixture with plasmid 10 μl, NdeI 1 μl, BamHI 1 μl, 10X NEB buffer 2 μl and sterile water 6 μl. The pET28a vector was restricted by enzymes Ndel and BamHI to generate cohesive ends. The reaction mixture contained 10 μl of IpET28a vector, 1 μl of Ndel, 1 μl of BamHI, 2 μl of 10X NEB buffer and 6 μl of sterile water. Both reactions were incubated at 37°C for 2 hours. Pass gene fragments through gel elution kit Purified and ligated into pET28a vector. It was further transformed into a propagation host E. coli TOP10 cell to propagate the linked plasmid. Such plasmids w...
Embodiment 2
[0138] Example 2: Construction of plasmid pET28aULL2INS
[0139] The gene encoding proinsulin along with the nucleotide sequence of SEQ ID NO: 10 encoding the peptide ULL2INS was designed, codon optimized and chemically synthesized, and cloned in pUC57 to make pUC57ULL2INS. The gene fragment was cloned into pET28a vector. Restriction digestion of the pUC57ULL2INS plasmid was performed by setting up a reaction mixture with 10 μl plasmid, 1 μl NcoI, 1 μl BamHI, 2 μl 10X NEB buffer and 6 μl sterile water. The pET28a vector was restricted by enzymes NcoI and BamHI to generate cohesive ends. The reaction mixture contained 10 μl of pET28a vector, 1 μl of NcoI, 1 μl of BamHI, 2 μl of 10X NEB buffer and 6 μl of sterile water. Both reactions were incubated at 37°C for 2 hours. Pass gene fragments through gel elution kit Purified and ligated into pET28a vector. It was further transformed into a propagation host E. coli TOP10 cell to propagate the linked plasmid. Such plasmids w...
Embodiment 3
[0140] Example 3: Construction of plasmid pET28aULL1LSP
[0141] To obtain construct pET28aULL1LSP, PCR-based site-directed mutagenesis was performed in plasmid pET28aULL1INS. Site-directed mutagenesis will change the B28 and B29 positions of the B chain from PK to KP. Use the following mutant primer pairs
[0142]Forward: 5'GTG GTT TCT TTT ATA CCA AAC CGA CCA AAC GTG GCA TTG T 3'
[0143] Reverse: 5'ACA ATG CCA CGT TTG GTC GGT TTG GTA TAA AAG AAA CCA C 3'
[0144] The PCR reaction mix consisted of 300 μM dNTP mix, 1X PFu buffer, 10 pm of each primer, 1 μl template plasmid and 41 μl sterile water. The PCR conditions used were: 94°C-8min, 94°C-40s, 55°C-40s, 68°C-3min (20 cycles) and 68°C for 10min. The site-directed mutagenesis product was digested with DpnI, and then transformed into the propagation host Escherichia coli TOP10 cells for propagation. Use the plasmid The miniprep kit was isolated and then transformed into E. coli Gold BL 21DE3 cells for protein expressio...
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