Genetic engineering production of human growth hormone
Abstract: Human growth hormone (HGH) is a non-glycosylated peptide hormone that stimulates body growth and has important clinical applications. The source of HGH is very limited. It can only be extracted from the pituitary gland of human corpses at first, which is far from meeting the requirements of clinical medication. Modern genetic engineering brings vitality to the production of this hormone, and produces a large amount of recombinant human growth hormone (rHGH) by mass culture of microorganisms in vitro. This paper focuses on the main technical routes for the production of rHGH by recombinant Escherichia coli.
Keywords: recombinant human growth hormone (rHGH) Escherichia coli fermentation
Scientists have found that human growth stimulating (HGH) is a non-glycosylated protein hormone secreted by the anterior pituitary gland. Natural HGH is a single-stranded spherical acidic protein (isoelectric point 5.2) with a total of 191 amino acid residues. The molecular size is about 22KD, contains two pairs of disulfide bonds, is not glycosylated, and forms a dimer through a disulfide bond between the chains. In-depth scientific research indicates that the Aa peptide of N-terminal 1 to 134 of HGH is an essential active sequence, and the C-terminal peptide acts to stabilize HGH molecules and prevent HGH from being degraded by proteases in the blood; HGH is an exocrine protein, nascent peptide chain For the precursor of active hormone, it needs to be activated by excision of the signal peptide .
Human growth hormone has an important role in promoting bone, visceral and systemic growth, activating protein biosynthesis, affecting fat and mineral metabolism, and is one of the key hormones in the body’s development [2,3]. At present, HGH is mainly used in adolescents for short stature, postoperative and severe burns, heart failure, anti-aging, etc. . With the aging of the population and the increasing number of patients with cardiovascular disease, the application of rhGH in the treatment of cardiovascular diseases is becoming more and more important. In addition, it has been reported that long-term injection of small doses of HGH (0.81 U / d) can delay the aging of various organ systems, which is an increase in the average life expectancy of people by 20 years. At present, human growth hormone drugs are far from meeting the needs of clinical drugs. Due to the development of biotechnology, recombinant human growth hormone (rHGH) has been successfully produced by using genetic recombination technology, and rHGH can be produced in large quantities by fermentation engineering. The following is a focus on the technical route to produce rHGH by genetic engineering.
1. Process development overview
The preparation of human growth hormone has undergone two stages of natural extraction and fermentation production of genetically engineered bacteria. The former has been eliminated due to resource limitation and extraction efficiency of growth hormone, and the latter has undergone two generations of development. The first generation of rHGH was directly expressed in E. coli, resulting in the recombinant protein Met-HGH, a methionine at the N-terminus than native HGH, which produced immunogenicity ; and several amino acids attached to the amino terminus were required. Degradation in vitro, after resection can become natural HGH .
The second generation of rHGH is a secretory expression type, and the signal peptide of the HGH precursor is automatically excised by a specific signal peptide cleavage enzyme on the inner membrane of the E. coli receptor during secretion to produce a recombinant Phe-HGH which is consistent with the native protein sequence. At the same time, the immunogenicity brought by Met is eliminated [2, 4]. In addition, the use of E. coli and the production of rHGH using eukaryotic microorganisms (including animal cells) has a shorter production cycle and a significantly higher yield. Because of these advantages, the production of recombinant Phe-HGH by E. coli has become the mainstream process for the production of genotropin HGH.
2. Genetic engineering production of rHGH process
2.1. Construction, screening and identification of rHGH engineering bacteria
The HGH full-length gene was first cloned. According to the amino acid sequence of human growth hormone, the codons preferred by E. coli were selected, and a small fragment containing 20 oligonucleotides was artificially synthesized, and the full-length sequence of HGH gene was synthesized by overlapping region PCR amplification. The OmpA signal peptide sequence was added to the structural gene, the NdeI restriction site was designed at the 5′ end, the stop codon TATAGGA was added to the 3′ segment, and the BamHI restriction site was designed .
Then, the HGH and PIN-III-OmpA plasmids were separately digested, and the recombinant Phe-HGH expression vector was synthesized under the action of buffer environment and DNA ligase (Fig. 1), and the recombinant molecule was amplified by PCR .
The logarithmic Escherichia coli was treated with calcium chloride hypotonic solution in ice bath to become competent cells. The recombinant plasmid was added to form a complex with calcium chloride and adhered to the surface of bacterial cell membrane, and then treated with heat shock at 42 °C. The recombinant plasmid enters competent E. coli due to increased cell membrane permeability .
Figure-1 Recombinant Phe-HGH expression vector .
The recombinants were screened by single cell cloning and detection of recombinant expression products, and the production was identified from the tissues, and the fermentation production conditions were explored and optimized by the two-sided response method. Finally, the inclined culture method is used to cryopreserve the engineering strain.
2.2. Fermentation production rHGH
The strain is first activated, expanded by a seed tank, and then fermented in a fermentor. The culture conditions are closely monitored during the cultivation process and adjusted as appropriate. The main parameters of fermentation culture are controlled as follows :
(1) The seeds are cultured overnight, and the 0D value is preferably in the range of 2 to 3.
(2) The fermentation culture time is generally 16-18 h, and the culture temperature is 37 °C.
(3) Maintain a pH of 7. during the fermentation. O±0.5, dissolved oxygen is not less than 20%.
(4) In the process of cultivation, the flow acceleration of glucose and trace amount should be adjusted according to the growth condition to keep the dissolved oxygen not less than 20%.
(5) The OD600 value of the fermentation body should be around 70, and the expressed rHGH should be more than 15% of the total body protein.
The fermentation production process is shown in Figure-2:
Figure-2 rHGH API production process 
2.3. Separation and purification of rHGH
2.3.1 rHGH rough lifting
First, the engineering cells were separated, and the culture solution was collected, precipitated at the isoelectric point of pH 5.5, salted out by ammonium sulfate, and collected by centrifugation .
2.3.2 Chromatographic purification of rhGH
The rHGH protein was purified by G-25 chromatography, Phenyl-Sepharose FF chromatography, Sephacryl S-100 chromatography and DEAE-Sepharose FF ion exchange chromatography to obtain residual DNA, residual engineered protein, related proteins and pyrogens. Relevant pharmacopoeia quality standards [7,8].
2.3.3 rHGH purity identification and activity identification
The purified sample was stained by non-reducing SDS-P AGE and chrome-silver method, and the purity was determined by thin layer scanning. The purified rHGH sample was taken and the protein activity was determined by HPLC (required specific activity was greater than 2.5 IU/ml)  .
The use of genetic engineering and fermentation engineering to produce human growth hormone (rHGH) is an effective way to increase production speed and production efficiency, can expand production, and basically alleviate clinical drug tension. However, the demand for drugs is still growing, and the prices of production plants and drugs are still high. Therefore, it is necessary to strengthen scientific research and increase the production volume by increasing the level of technology.