Secretory Expression Of Human Growth Hormone In Pichia Pastoris

Recombinant human growth hormone?rhGH?is a protein necessary for human growth and development.It is very important for human growth and development,so it is in great clinical need.Therefore,it is necessary to develop an efficient fermentation process to produce rhGH with high purity.In order to obtain high expression of recombinant human growth hormone in Pichia pastoris,According to the amino acid sequence and codon preference of E.coli,the human growth hormone gene were optimized to avoid the rare codons.Meanwhile,in its upstream added a promoter and a signal peptide sequence,so the total length of the synthesis gene is 1040 bp,designed and synthesized 30 oligonucleotides DNA fragments.The whole DNA sequence was synthesized by improved two-step method,and then inserted to pUC18 vector.Positive clones identified by blue-white colony screening was sended to sequence.The result showed that two clones was correct completely in all four positive clones.The growth hormone gene was obtained by PCR amplification technology,and then inserted into Pichia pastoris.In the Pichia pastoris expression vector pPIC9K,which contains the AOX1 promoter and?secretion signal peptide sequence,a pPIC9K/hGH recombinant yeast expression vector was constructed,and then Pichia pastoris GS115 was transformed by electric shock.The strain was screened by G418 and integrated With multiple copies of the human growth hormone gene,the gene strain was successfully cultured in shake flasks and induced by methanol to successfully achieve the secretion and expression of growth hormone in Pichia pastoris.Activity analysis was then performed using SDS-PAGE technology.The amplification of yeast genomic DNA by polymerase chain reaction showed that the target gene had been successfully integrated into the chromosome of Pichia pastoris.The selected strains used methanol to induce the expression of the recombinant target protein.SDS-PAGE and Western Blotting technology were used to identify the expressed products.There was a very obvious band near 22kD,and the color became darker and deeper with the increase of induction time.However,no bands appeared in the control group.The results of Western blotting indicated that the recombinant protein was recombinant human growth hormone.The standard concentration of casein was determined by the Folin-phenol method,and the total protein content in the supernatant was measured to be 1.21mg/ml.The OD₄₉₀ value of hGH measured by the ELISA method was 0.217,calculated by the formula of the standard curve,the content of hGH in the supernatant was 330?g/ml.From this calculation,hGH accounts for 27.2%of the total protein in the supernatant.When performing the first step chromatography on the sample after 3K ultrafiltration,select Q-sepharose FF as the chromatography medium,10mM PB,pH7.5 as the loading buffer,and use 10%Solution B to elute the target protein;For two-step chromatography,select phenyl-sepharose FF as the chromatography medium,10mM PB+1M?NH₄?₂SO₄,pH7.5 as the loading buffer,and use 10mM PB,pH7.5as the elution buffer,after two steps After chromatography,the purity of rhGH reaches more than 98%.The matrix-assisted laser desorption ionization time-of-flight mass spectrometry?MALDI TOF MS?determined that the relative molecular weight of the recombinant hGH was 22 113 Da,and the N-terminal amino acid sequencing showed that the 7amino acids at the N-terminus of rhGH were PTIPLSR.The biological activity of the recombinant hGH was measured by the pituitary gland rat weight method,and the experimental reliability test met the standard.The PT of the same batch of rhGH samples was 4.29IU/1.33mg,representing the confidence limit rate?FL?of the test error?FL?of 26.6%.Live is 3.2 IU/mg.In addition,the fermentation process of rhGH has been optimized,which can greatly improve the expression level of rhGH protein.In summary,this experiment provides an effective fermentation process for the production of high-purity human growth hormone,which can reduce the production cost of large-scale production of human growth hormone,and establish a platform of Pichia pastoris expression system that efficiently expresses human growth hormone,for reorganizing life The industrial production of long hormones has laid the foundation.