The Role Of Different Promoters Of Pichia Pastoris And Its Application In HPV16 L1 Expression

Objective To investigate the expression characteristics of heterologous protein controlled by different promoters in Pichia pastoris under different cultivation and induction conditions; to investigate the effects on HPV16L1expression of the combined application of diffrerent promoters-driven expression units; in addition, to develop a universal plasmid system and assess its potential for shuttling expression between P. pastoris and Hansenula polymorpha. The study sought to optimize HPV L1expression from perspective of the use of promoters with distinct features.Methods The oligonucleotide fragments of constitutive translation elongation factor1-a promoter (PTEF1) and formaldehyde dehydrogenase promoter (PFLD) were amplified from P. pastoris GS115by PCR. Recombinant plasmids were constructed by replacing the alcohol oxidase1promoter (PAOX1) of commercially derived expression vector pPink-HC and pPink-LC with PTEF1and PFLD, and cloning the genes of green fluorescence protein (GFP) and HPV16L1protein to downstream of the promoters respectively. The heterologous protein expression levels driven by PTEF1in different carbon sources (glucose、glycerol、methanol、sorbitol) and the correlation of cell growth state with utilization of different carbon sources were checked. The experession characteristics of GFP and HPV16L1controlled by PFLD were investigated with the use of different carbon and nitrogen sources. By constructing an expression pasmid carrying PAOX1or PTEF1-directed two seperated expression units or introduing two expression units into one host cell by mutiple transformation, the influence of combined application of different promoters on improving HPV16L1expression level were studied. Furthermore, by constructing a universal expression vector with rDNA of Hansenula polymorpha as an intergartion element and promoters from H. polymorpha and P. pastoris to direct the gene expression, gene integration efficiency into yeast chromosome and protein expression levels mediated by the related elements in the universal plasmid were discussed.Results Under culture conditions with glucose (Glu) or methanol (M) as carbon source, PTEF1directed successful expressions of GFP and HPV16L1at24h after ionculation, and subsequently the expression level declined sharply. However, in media with glycerol (Gly) or sorbitol (Sor) as carbon source, a sustained efficient expression was obtained. P. pastoris cells have a faster growth rate in media with Glu or Gly. In the four induction media composed of Sor+NH4+、Sor+MA、M+NH4+and M+MA, efficient expressions of HPV16L1were evidenced at24h、48h、72h after induction. The highest expression level was obtained in M+NH4+and M+MA media, and Glu+NH4+, Glu+MA, but Gly+NH4+, and Gly+MA media only showed fewer or merely detectable protein expression. The expression units controlled by the PAOX1or PTEF1successfully directed co-expression of GFP and/or HPV16L1, but protein levels decreased in comparsion with that obtained with a single expression unit. The rDNA and autonomously replicating sequence (ARS) elements from H. polymorpha mediated transformation in both yeat hosts, but the efficiencies of transformation and integration were low in P. pastoris. Through a screening combined with multiple passages under non-resistant pressure and resistant selection, the plasmids containing the H.polymorpha-rDNA stably integrated into Pichia pastoris genome. The H.polymorpha Pmox and P.pastoris PAOX1and PTEF1worked in both hosts. The universal plasmid vector mediated efficient protein expression of GFP and HPV16L1in both P.pastoris and H. polymorpha.Conclusions Heterologous proteins were successfully expressed in P. pastoris with the uses of different promoters. The different culture conditions produced significant influences on promoter function and protein expression. Co-expession of different promoters-driven expression units were confirmed, but showing an interference to each other. Constructed uiniversal plasmid vector mediated shuttling expression of herterogeneoius protein in P. pastoris and H. polymorpha. From the perspective of appropriate application of promoter, this current study provides an optimization strategy of efficient expression of heterologous gene in yeasts. And, successful expression of HPV16L1in P. pastoris obtained by using different promoters provides an optional preparation platform for the development of a low-cost multivalent HPV vaccine.