Study On Expression Of Human Epidermal Growth Factor In Pichia Pastoris

Human epidermal growth factor is a single polypeptide chain composed of53 amino acid residues with a molecular weight of about 6000Kda,which has excellent stability.Human epidermal growth factor has the functions of promoting the growth,differentiation of epithelial cells,repairing damaged mucosa and forming a stratum corneum to protect the skin,etc.However,due to fewer sources and higher purification costs,human epidermal growth factor is limited in industrial production and cannot meet the market demand.In this study,the effects of sequence optimization of signal peptide,gene dose,co-expression of PDI and transcription factors Aft1 and Hac1p on rhEGF expression were studied in order to obtain the engineered P.pastoris with high rhEGF expression.(1)According to pichia pastoris codon preference,the signal peptide was optimized and the optimized sequence N?was inserted into p PICZ?A to obtain recombinant vector p PICZ-N?.Using p PICZ-N?and p PICZ?-rhEGF as templates,two fragments,N?and rhEGF were obtained.The recombinant fragment N?-rhEGF was prepared by SOE-PCR,and was inserted p PICZ?A to obtain p PICZ-N?-rhEGF.The recombinant vectors were transferred into Pichia pastoris KM71 genome,the expression strains KM71-p PICZ-?-rhEGF and KM71-p PICZ-N?-rhEGF were obtained.Strain KM71-p PICZ-?-rhEGF and KM71-p PICZ-N?-rhEGF were induced by methanol for 96h in shake flask,and the rhEGF expression levels were a nalyzed.The total amount of protein and the total biological activity of KM71-p PICZ-N?-rhEGF arrived at 134.5165±17.7482 mg/L and 141242±18128 IU/m L,respectively,which were increased by 44.22%and 47.02%,respectively,compared with KM71-p PICZ?-rhEGF.(2)Starting from plasmid p PICZ-N?-rhEGF(containing 1 copy of rhEGF expression cassette),a series of recombinant vectors,p PICZ-2N?-rhEGF,p PICZ-3N?-rhEGF and p PICZ-4N?-rhEGF,p PICZ-5N?-rhEGF with 2?3?4 and5 rhEGF expression cassette,respectively,were constructed through using same tail enzymes Bam H?and Bgl?,and link His4 fragments.The linearized recombinant plasmids p PICZ-2N?-rhEGF-His4?p PICZ-3N?-rhEGF-His4?p PI CZ-4N?-rhEGF-His4 and p PICZ-5N?-rhEGF-His4 were transferred into Pichia pastoris KM71 by electroporation.As a result,recombinant strains KM71-p PICZ-N?-rhEGF?p PICZ-2N?-rhEGF-His4?p PICZ-3N?-rhEGF-His4?p PICZ-4N?-rhEGF-His4?p PICZ-5N?-rhEGF-His4 were obtained,containing 1?2?3?4and 5 copies of rhEGF expression cassette,respectively.Strains KM71-p PICZ-N?-rhEGF?p PICZ-2N?-rhEGF-His4?p PICZ-3N?-rhEGF-His4?p PICZ-4N?-rhEGF-His4?p PICZ-5N?-rhEGF-His4 were induced by methanol for 96h in shake flask.The total amount of extracellular protein s ecreted by the five strains arrived at 130.7635±14.6523 mg/L?143.9850±17.862 mg/L?169.6153±17.6628mg/L?237.6390±23.6773mg/L?127.3843±15.6234mg/L,respectively.The total biological activity of fermentation supernatants by the five strains arrived at 137040±25124 IU/m L?153344±23105 IU/m L?175551±19862 IU/m L?235052±23398 IU/m L?1118467±24170 IU/m L,respe ctively.Results show that strain KM71-p PICZ-4N?-rhEGF-His4 had the highest extracellular protein and biological activity,the total amount of protein and the total biological activity were increased by 81.73%and 71.52%,compared with strain KM71-p PICZ-N?-rhEGF.The total amount of protein and the total biological activity by strain KM71-p PICZ-5N?-rhEGF-His4 were only 53.60%and 50.40%,respectively,as compared with those of KM71-p PICZ-4N?-rhEGF-His4.(3)The recombinant plasmids p PIC9K-PDI-G418,p PIC9K-Aft1-G418 and p PIC9K-Hac1p-G418 were transferred into KM71-p PICZ-4N?-rhEGF-His4 genome by electroporation and the co-expression strains KM71-p PICZ-4N?-rh E GF-His4-PDI?KM71-p PICZ-4N?-rhEGF-His4-Aft1 and KM71-p PICZ-4N?-rh EG F-His4-Hac1p were obtained.Strains KM71-p PICZ-4N?-rhEGF-His4?KM71-p PICZ-4N?-rhEGF-His4-PDI?KM71-p PICZ-4N?-rhEGF-His4-Aft1 and KM71-p PICZ-4N?-rhEGF-His4-Hac1p were induced by methanol for 96h in shake flask.The total amount of extra cellular secreted protein arrived at 227.4239±20.1687 mg/L?144.5416±20.0676 mg/L?171.006±24.3572 mg/L?309.0315±25.4678 mg/L,respectively?the total biological activity arrived at 218554±27542 IU/m L?145987±24268 IU/m L?176136±25087 IU/m L?311812±22697 IU/m L.Hac1p co-expression had a significant effect on rhEGF expression.The total amount of protein;total biological activity of strain KM71-p PICZ-4N?-rhEGF-His4-Hac1p were higher than strain KM71-p PICZ-4N?-rhEGF-His4,which were increased by 35.88%and 36.68%,respectively.(4)The co-expressing strain KM71-p PICZ-4N?-rhEGF-His4-Hac1p was subjected to high-density fermentation in a 3L fermenter,and the total fermentation time was 96h.Finally,the OD_600nm and the total amount of protein of strain KM71-p PICZ-4N?-rhEGF-His4-Hac1p arrived at 240 and 1.6 g/L,respectively.