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RECOMBINANT HORMONE PRODUCTION In the human body, peptide hormones are secreted after encoding by peptide hormone genes in the specialized cells, for instance insulin and other Human Growth Hormone (hGH). INSULIN This peptide hormone is secreted by the b cells of Islets of Langerhans of pancreas which catabolises glucose in blood. Insulin is a boon for the diabetics whose normal function for sugar metabolism generally fails. Insulin consists of two polypeptide chains, chain A (21 amino acid long) and B (30 amino acid long). Its precursor is proinsulin which also contains two polypeptide chains, A and B, and is connected by a third peptide chain C (35 amino acid long). However, the recent discoveries reveal that precursor of insulin is pre-proinsulin which is about 109 amino acid long. The pre-proinsulin is synthesized in b cells of Islets of Langerhans of pancreas, the structure is given below; NH2 (Peptide) B chain (Peptide C) A chain COOH Efforts were made to isolate mRNA for preproinsulin. Thereafter, it was inserted into a plasmid like pBR 322. The recombinant plasmids were transferred into E.coli cells secreted pro-insulin. Itakura et al., (1977) chemically synthesised DNA sequences for two chains A and B of insulin and separately inserted into two pBR 322 plasmids by the side of b-galactosidase gene. The insulin gene and b-galactosidase gene are separated by a triplet codon for methionine. The recombinant plasmids were separately transferred into E.coli cells which secreted fused b-galactosidase A chain and b-galactosidase B chain separately. These chains were isolated by detaching the b-galactosidase gene by treatment with cyanogen bromide or trypsin which cleaves at methionine. Yield: 24 grams of healthy transformed cells can produce 10 mg of insulin (Sasson, 1984). It is estimated that clones of E.coli are capable of producing about one million molecule of insulin per bacterial cell. Human insulin (humulin) is the first therapeutic product produced by recombinant DNA technology by Eli Lilly & Co. SOMATOTROPIN Somatotropin, the hGH, is secreted by the anterior lobe of pituitary glands which consists of 191 amino acid units. Its secretion is regulated by two other hormones (somatostatin and growth hormone releasing hormone) produced by hypothalamus. Deficiency of somatotropin in about 3% cases is of heredity. It has been estimated to about I child in 5000. Turners syndrome is one of the most common chromosome disorders in girls and it is characterized by short stature and non-functioning of ovaries affecting approximately 1 in 2,500 live female births. The extraction of somatotropin pharmaceutically from the pituitary glands could not meet annual demand of this hormone. Biosynthesis of somatotropin was achieved through gene cloning procedures. Double stranded cDNAs were produced from mRNA precursor of hGH which was then incorporated into bacterial cells where it expressed in non precursor form. Bacteria were unable to convert peptide into biologically active form. A recombinant plasmid containing a full length hGH cDNA (which fails to express) is cleaved with restriction enzymes that release a fragment containing the complete hGH coding sequence after codon 24. Several overlapping complementary oligonucleotides are ligated to form one synthetic strand of small DNA fragment which contains the coding sequence for the first 24 amino acids of mature hGH (after removal of N-terminal signal peptide). The synthetic DNA and cDNA molecules are ligated to yield a new fragment which contains the complete coding sequence of hGH. It is ligated into a restriction site just down stream of the lac promoter / operater region cloned on a plasmid. The rDNA plasmids are allowed to transform E.coli. Synthesis of hGH is induced by an inducer of lac operon (Isopropylthiogalactoside IPTG). The hGH is subsequently purified. HGH is being commercially produced by this method. About 100,000 molecules of hormone per cell of E.coli have been produced (Newmark, 1979). One of the major difficulties was that at the N-terminal of polypeptide an extra methionine was attached and produces met-hGH. Methods are developed to remove methionine from the met-hGH molecules. SOMATOSTATIN Somatostatin, a 14 residue polypeptide hormone is synthesised in the hypothalamus. It is the first polypeptide which was expressed in E.Coli as part of the fusion peptide (Itakura et al., 1977) which inhibited the secretion of the growth hormone, glucagons and insulin. It does not contain any internal methionine. Eight single stranded DNA segments were synthesised chemically which were annealed in an overlapping manner to form a double stranded DNA (the synthetic gene). It had single stranded projections at the each end as the same are formed by EcoRI. The synthesised gene contained 51 base pairs which were terminated by two nonsense (stop) codons and precede by a methionine codon as below: ATG (42 basepairs encoding somatostatin) TGATAG. Two plasmids pSOM I and pSOM II-3 were constructed. The synthetic gene was introduced into E.coli b-galactosidase gene at different sites. The chemically synthesised gene was inserted downstream from the lac promoter in such a way that the gene fusion should have specified a polypeptide in which the first 7 amino acids of b-galactosidase were fused to somatostatin. But the somatostatin was not detected in transformed bacteria; possibly it was degraded in E.coli (Glover, 1994). An alternative plasmid, pSOM II-3, was constructed which had the both lac promoter / operater and lacZ regions. The lac Z gene encodes peptide of b-galactosidase. If the resulted frame of lac Z is maintained after inserting a DNA, a fusion peptide is produced. The plasmid was cleaved in the lac Z segment by restriction enzyme. The synthetic gene was inserted into the plasmid at EcoRI site near C-terminus of b-galactosidase. The plasmid in the transformed bacterium directs the synthesis of a fused protein consisting of NH2 terminal sequence of b-galactosidase fragment coupled by methionine to somatostatin. It was stabilized from proteolytic degradation by b-galactosidase moiety (Glover, 1984). This fused protein was purified and treated with cyanogens bromide (CNBr) which cleaves only protein at the carboxyl side of the methionine. Thus, the methionine linker remains attached to b-galactosidase fragment, and somatostatin was released. Now, it has become possible to inhibit the degradation of foreign protein in E.Coli by introduction of protease inhibition (PIN) gene of T4 phage. b-ENDORPHIN b-endorphin, 30 amino acid long neuro-peptide with opiate activity, is another growth hormone which was expressed in genetically engineered E.coli cells. Shine et al., 1980, integrated DNA sequences of b-endorphin, obtained from mRNA, adjacent to b-galactosidase genes on plasmid. The mRNA contained large precursor of protein that consisted of, besides b-endorphin, the hormones a-melanotropin, corticotrophin, b-lipotropin and b-melanotropin. The b-endorphin is cleaved from C-terminus of the precursor peptide. In this way, the transformed bacteria produced an insoluble fusion protein between b-galactosidase and b-endorphin. b-endorphin can be removed from the hybrid protein by trypsin which cleaves only at arginine residue. Before doing so, internal lysines are protected from trypsinization by citraconylation as below; NH3 - b-galactosidase -b-melanotropin-b-endorphin-COOH || b-galactosidase + b-endorphin
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