Production of human growth hormone (hGH) by recombinant DNA technology
- The gene for human growth hormone (hGH) is isolated from human pituitary gland.
- Insertion of whole hGH gene into plasmid vector and cloning into E.coli results into production of biologically inactive hormone because bacteria can translate the region of gene that are not translated in human thereby producing a prehormone containing an extra 26 aminoacids which might be difficult to remove.
- Hence the segment of gene that codes for the first 24 aminoacids of hormone is constructed chemically from blocks of nucleotide.
Step I: Chemical synthesis of gene for first 24 aminoacids:
- From the known aminoacids sequence of hGH, gene for first 24 aminoacids are constructed chemically. These genes are constructed in three small fragments and then they are joined by T4 DNA ligase to get whole gene for first 24 aminoacids.
Step II: Isolation of mRNA for hGH
- In this step mRNA for hGH is isolated from human pituitary gland tissue.
Step III: Reverse transcription
- Using reverse transcriptase enzyme complimentary DNA (cDNA) is synthesized from mRNA.
- The cDNA obtained by reverse transcription process, is the gene for hGH.
- The full gene is cut with restriction endonuclease enzyme to remove first 24 gene.
Step IV: Joining of synthetic gene and cDNA
- In this step synthetic gene (gene for first 24 aminoacids) and cDNA are joined in order to obtain full gene with its own initiation codon (AUG). T4 DNA ligase join these genes.
Step V: selection of suitable vector and recombination:
- Expression vector phGH407 derived from plasmid vector PBR322 is used as carrier vector.
- HGH gene is ligated into a restriction site just downstream of Lac; promotor/operator region of the expression vector.
Step VI: selection and recombination into suitable host cell
- E. coli is used as suitable host cell.
- The recombinant expression vector is then transformed into E.coli.
- The recombinant E. coli then starts producing hGH.
- The recombinant E. coli are isolated from the culture and mass production by fermentation technology to obtain hGH.
Use of recombinant human growth hormone (hGH)
- Treatment of children suffering from growth deficiency
- Treat the patient with Tumer’s syndrome and chronic renal insufficiency
- To treat patient with renal carcinoma
- Bovine somatotropin hormone is used to increases milk production in lactating cows and also to increase body mass of cattles.