Expression,purification And Activity Of Recombinant Human Growth Hormone And Its Mutants

Human growth hormone(hGH)is an important protein hormone secreted by the anterior pituitary of the hypothalamus.It plays a key role in various life activities such as cell proliferation and metabolism.It is mainly used to treat dwarfism.Currently,recombinant human growth hormone(rhGH)was approved.But the price was very high.Furthermore,there are various defects for hGH,such as poor compliance,low bioactivity,and allergy.Therefore,obtaining rhGH with high expression and high activity is of great significance for reducing industrial production costs and improving clinical efficacy.Because of the current problems,this thesis has carried out the following four aspects of research work focusing on hGH.1.Genetic screening of growth hormoneWe conducted a preliminary genetic screening of growth hormone from the mouthwash samples of some people.The results showed that the base sequences of the exon part of growth hormone in the samples were the same,suggesting that human growth hormone gene sequences might be relatively conservative.Therefore,it was of great significance to biosynthesize growth hormone and perform exogenous supplementation.2.Cloning,expression,purification,and structural characterization of recombinant human growth hormone in E.coliIn order to increase the expression of rhGH in vitro,we inserted a guide peptide sequence at the N-terminus of the hGH gene.A secreted expression strain of rhGH wa successfully constructed and soluble expression was achieved.After optimizing the culture conditions,rhGH with an expression level of 800 μg/m L was obtained,and its purity can reach over 90%.The obtained rhGH was analyzed by SDS-PAGE,Western blot,HPLC,CD and protein mass spectrometry,which proved the expressed protein was consistent with the structure of natural human growth hormone.3.Expression,purification and structural characterization of growth hormone mutants in E.coliIn order to obtain highly active rhGH,we conducted site-directed mutations at the key amino acids in the three-dimensional structure of rhGH.Six mutant genes were successfully cloned and six mutant proteins of growth hormones were obtained.The results of gene sequencing,western blot and protein mass spectrometry analysis showed that the mutant protein sequences were correct and the mutations were successful.4.Bioactivities of recombinant human growth hormone and its mutantsWe used zebrafish and cell models to evaluate the activities of wild-type and mutant growth hormones.Compared with the wild type,mutants F176 Y and Q46 W have stronger growth-promoting effects on zebrafish internode blood vessels and sub-intestinal blood vessels.Furthermore,they have stronger growth-promoting effects on MC3T3-E1 cells.Conclusion:This thesis analyzes that the human growth hormone gene sequence may be relatively conservative,suggesting that supplementing exogenous growth hormone has important significance,at the same time,a simple and effective method was reported for cloning,expression,and purification of rhGH in E.coli.This study successfully expressed the wild-type rhGH and its six mutants in the periplasm of E.coli.The mutants F176 Y and Q46 W showed strong biological activity,suggesting that this site may be a key site for the hGH activity.This study provided the foundation for the industrial production of rhGH and its clinical applications.