Human growth hormone hGH is a single-chain polypeptide that participates in a wide range of biological functions such as metabolism of proteins, carbohydrates and lipids as well as in growth, development and immunity. Growth hormone deficiency in human occurs both in children and adults. The routine treatment for this condition is administration of recombinant human growth hormone rhGH made by prokaryotes.
Since nonglycosylated human growth hormone is a biologically active protein, prokaryotic expression systems are preferred for its production. Different strains of E. High levels of rhGH were produced using E. This simple and cost effective production process could be recruited for large scale production of rhGH. Human growth hormone hGH is a single-chain polypeptide hormone mainly synthesized by the acidophilic somatotrophs of the anterior pituitary gland.
Growth hormone GH has important effects in stimulating the metabolism of bone, cartilage and muscle,[ 15 ] and somatic growth during childhood. Cardiomyopathy, decrease in myocardial mass with systolic and diastolic dysfunction both at rest and during exercise, has been reported in GH deficiency GHD adults. It was reported that GHD adolescents had reduced diastolic filling, cardiac size and disordered cardiac function. Until the mids, the only source of hGH was from human cadaver tissue.
The use of pituitary-derived hGH was prohibited when its association with Creutz feldt-Jakob disease was proved. Recombinant DNA technology has facilitated a safe and abundant production of rhGH in various heterologous systems, without risk of transfer of human pathogens, eliminating the requirement for pituitary-derived preparation.
Recombinant hGH rhGH is now largely used to treat GH deficient short-stature children to final height and as therapy of adults with GHD,[ 1 ] acceleration of wound healing, as well as an increase in insulin-like growth factor IGF -1 levels in the blood from low to normal.
In the present study, over-expression of rhGH under chemical induction in E. The rhGH production at different medium compositions and bacterial strains were compared and optimal medium and strain for over production of human growth hormone were defined. All experiments were repeated at least three times. For each strain, 10 ml of LB containing appropriate antibiotics was inoculated with a single recombinant E.
Induced T5 and non-induced T0 cells and media were harvested by centrifugation at rpm at room temperature. The cell suspension was then sonicated by applying 5 s on a medium intensity setting while holding on ice. For primary detection of production of rhGH by E. Membranes were washed three times with PBS containing 0.
Proteins were stained with Coomassie Brilliant Blue dye. Proteins were also transferred into a nitrocellulose membrane and primed with anti-GH antibody and continued as stated for dot blotting.
Between each step, wells were washed three times with washing buffer. Column was loaded with samples containing rhGH and then washed with 5 ml binding buffer, followed by washing with 2. The specificity and molecular weight of the rhGH was confirmed by Western blotting method using a specific monoclonal antibody against hGH. Transformation efficiency and plasmid stability were assessed. The Top10 strain proved to be the best strain for transformation with this plasmid.
Comparison of transformation efficiency between different E. Cells were harvested 5h after induction of protein production by IPTG and rhGH production was assessed in the samples. Dot blot analysis showed that all strains have the ability to produce human growth hormone as it was detectable in both cell lysate and medium [ Figure 2 ]. Dot blot analysis of E. Top10 none transformed supernatant, A9: Top10 none transformed cell lysate, A Top10 transformed LB T0 supernatant, A Top10 transformed LB T0 cell lysate, B1: Top10 transformed LB T5 supernatant, B2: Top10 transformed LB T5 cell lysate, B4: Top10 transformed 4YT T0 supernatant, B6: Top10 transformed 4YT T0 cell lysate, B7: Top10 transformed 4YT T5 supernatant, B8: Top10 transformed 4YT T5 cell lysate, B Top10 repeat 2, D1 to E1: Based on sequence analysis of the inserted gene, molecular weight of rhGH was expected to be around 27 kd.
This subject was confirmed by Western blotting. Subsequently, ELISA technique was used to quantify and compare the levels of rhGH produced by different strain in order to determine the best protein producing strain and culture conditions. Lane 1, molecular weight marker; Lane 2, standard GH, Lane 3,4 bacterial lysate extract before purification; B Western blot analysis of bacterial lysate extract. The protein ladder has been highlited using pen marker for better visulalization.
Short stature in children due to growth hormone deficiency and chronic renal failure or Turner's syndrome is most often treated with human growth hormone. GH is one of the most widely used hormones in supplementation. Years of administration of this agent have proved its safety and efficacy in the therapy of various conditions associated with short stature.
Expression of recombinant protein in E. We produced rhGH in high concentration with a simple method and compared the ability of different strains of E. We extracted the protein from supernatant as well as inclusion body. However, some new problems rose, especially in case of inactive protein synthesis and inclusion body formation. Plasmid stability, expression of target protein in high level, chemical induction system, easy isolation of the inclusion bodies from cells, lower degradation of the expressed protein, high level of target protein homogeneity in inclusion bodies, simple purification and possibility to reduce the number of purification steps are usually indicated as the main advantages of inclusion body formation.
Six histidine residues in the fusion partner facilitate the purification of the fusion protein by the highly selective metal affinity chromatography. The inducible promoter systems, such as trc, are advantageous for overproducing recombinant proteins in the bacteria. Separation of the two phases is especially useful when optimal growth conditions are not conducive for optimal product formation, which is frequently encountered in production of recombinant proteins from genetically engineered micro-organisms.
In fed-batch cultures, the E. In conclusion, as shown in this article, high levels of rhGH production can be achieved in prokaryotic host in an inducible manner. The high expression level, affinity purification, and the maintenance of high expression level in the high density cell culture allow producing large quantities of rhGH which will enable us to produce this important hormone for therapeutic uses and a good source of hGH for further scientific investigation.
Normal flora bacteria can also be used for in situ production of rhGH in human body. The efficiency and safety of later method need further investigations. If proven to be effective, controllable and safe, this approach could be applied to other therapeutically important proteins which will result in a dramatic reduction in cost of treatment.
National Center for Biotechnology Information , U. J Res Med Sci. Marzieh Rezaei and Sayyed H. Iran Find articles by Sayyed H. This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-Share Alike 3.
This article has been cited by other articles in PMC. Media, Cultivation and Expression: Open in a separate window. Characterization of Produced rhGH Based on sequence analysis of the inserted gene, molecular weight of rhGH was expected to be around 27 kd. Footnotes Source of Support: Nil Conflict of Interest: Sereikaite J, et al.
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