Insulin is a secreted protein produced by eukaryotic cells, and its synthesis and secretion require the direct participation of the endoplasmic reticulum and the Golgi apparatus in order to process and modify the synthesized protein, and obtain insulin with a certain spatial structure and biological activity. E. coli, on the other hand, is a prokaryote that does not have an endoplasmic reticulum and Golgi bodies and cannot process and modify synthetic proteins. So how does E. coli that introduces the insulin gene produce insulin with biological functions?
There are three main construction schemes for the production of human insulin E. coli engineering bacteria:
1. AB chain separate expression method
This method is used in the early recombinant production of insulin by E. coli. The gene-coding regions of the A and B-chains (without introns) are chemically synthesized and cloned, respectively, on the expression-type plasmids of the β-galactosidase gene, which forms a heterozygous gene with the insulin coding sequence, and its junction is a methionine codon. The recombinant molecules transform E. coli receptor cells, and the two clones synthesize β-galactosidase-human insulin A-chain fusion protein and β-galactosidase-human insulin B-chain fusion protein, respectively. After large-scale fermentation, the fusion protein is isolated and purified from the bacterium, and then the fusion protein is chemically cut off by cyanide at the C end of the methionine residue, releasing the A and B chains of human insulin. Since β-galactosidase contains multiple methionine residues, cyanide bromide is treated to generate multiple small molecule polypeptides, while the A and B chains do not contain methionine residues, so they are not further degraded by cyanogen bromide. After further purification of the A and B chains, they are mixed in a molecular ratio of 2:1 and chemically folded in vitro.
2. Human insulin expression method
The human proinsulinogenic cDNA coding sequence is cloned downstream of the β-galactosidase gene, and the junction of the two DNA fragments remains methionine codons. After the heterozygous gene is efficiently expressed in Escherichia coli, the fusion protein is isolated and purified, and the human proinin insulin fragment is recovered by cyanide-specific chemical cleavage, and then folded in vitro. Due to the presence of C-chains, proinsulin can form a natural spatial conformation under renatural conditions, providing good conditions for the correct pairing of three pairs of disulfide bonds, so that the folding rate in vitro is as high as 80%. In order to obtain biologically active insulin, the folded human insulin proindomol must be specifically excised with trypsin. The site of action of trypsin is located at the carboxyl end of arginine or lysine residue, due to the presence of natural conformation, arginine residues at position 22 of the human insulin proprochain and lysine residues at position 29 are not sensitive to the action of trypsin. Therefore, after treating human proinsulin with trypsin, a complete A-chain and a B-strand with arginine residues at the C-terminus can be obtained. That is, compared with the natural insulin of man, this B chain has an additional amino acid residue, which is then specifically excised with a high concentration of carboxypeptidase B.
3. AB chain simultaneous expression method
The basic idea of this method is to stitch together the gene coding sequences of the A and B chains of human insulin, and then assemble the downstream of the E. coli β-galactosidase gene. After the fusion protein expressed by the recombinant is treated with cyanide bromide, the A-B chain polypeptide is isolated and purified, and then the A-chain and B-chain peptides are obtained by corresponding cleavage methods according to the amino acid residue properties at the junction of the two chains, and finally the active recombinant human insulin is prepared by in vitro chemical folding. Similar to the AB chain expression method, its biggest drawback is still the low accuracy rate of in vitro folding.
E. coli cannot express biologically active insulin because E. coli can only express insulin genes in cells without specific spatial structures, and because they lack the protein processing system of eukaryotic cells. The construction strategies of the above three kinds of engineered bacteria all adopt the method of splicing insulin gene and E. coli β-galactosidase gene, and the fusion recombinant protein produced cannot be secreted, mainly in the form of inclusion bodies, and then the peptide chain is isolated, purified, and folded in vitro to obtain biologically active insulin.