Optimized Expression Of Human Papillomavirus Major Capsid Proteins

Objective To investigate the influence of multi factors in E.coli and Pichia pastoris on expression of high-risk human papillomavirus (HPV) L1 proteins and lay a foundation of development of low-cost and abroad vaccines against multi-genotypes of HPV.Methods on the basis of prokaryotic expression, recombinant plasmids for non-fusion and different fusion forms of HPV L1 genes were constructed, including HPV L1 sequence of different genotypes and the L1 sequence of identical genotype and different characters, and transformed to various host bacteria(E.coli DH5a and E.coli BL21), and induced with 1PTG at various temperatures(37℃ and20℃). The fusion proteins were analyzed for expression and solubility by 12% SDS-PAGE, and identified by Western blot. Besides, two optimized versions of the gene that encodes HPV 16 L1 (M16 and Y16), which were designed according to the codon usage frequency of Pichia pastor is, a wild type (W16), and a version optimized for expression in mammalian cells (P16) were used for assessing expression in Pichia pastoris. All gene sequences were analyzed for mRNA secondary structure by simply calculating CAI (codon adaption index), free energy and GC contents. Furthermore, the use of deficiency of proteinase in engineered host strains and plasmid vector pPink HC which theoretically guarantees high gene copy numbers in the selected colonies and pPink LC were all used. In addition, the recombinant proteins with N-terminal fusion of Glutathione S-transferase or alpha factor from Saccharomyces cerevisiae, or with C-terminal truncation were all investigated to test the protein solubility, expression levels and VLPs assembly of HPV16L1 in P.pastoris.Results SDS-PAGE analysis showed that GST-HPV16 L1 fusion protein was efficiently expressed in E. coli DH5a, whereas there was no visible target protein expressed when either non-fusion or Trx-fusion protein strategies were used. There was no difference between the usage of host strains DH5a and BL21 for the expression of GST-fusion proteins. Almost all recombinant proteins produced in the form of inclusion bodies under induction at 37 ℃, whereas a large part of expressed fusion proteins when induced at 20 ℃ presented in soluble supernatants after ultrasonic and centrifugation treatment. The different sequence versions of HPV 16 L1 (including mammalian codon-optimized sequence, wild type and Pichia pastoris codon-optimized sequences) had little effect in terms of expression levels of fusion proteins. Gene sequences of HPV18 L1 and HPV58 L1 that codon-optimized according to the codon usage rates of yeasts were all efficiently expressed in E. coli cells in a form of GST-fusion protein, with a similar level to that of HPV16 L1. Besides, the expression of HPV16 L1 can be significantly improved in P. pastoris through optimizing codon usage. four gene sequences including M16,Y16,P16 and w16 among which M16 showedvthe highest expression efficiency, followed by Y16 and P16, whereas W16 was merely detectable. However, the codon adaptation order is sequentially Y16, M16, W16, and P16. Secondary structure of mRNA were simply analyzed by calculating free energy and GC%, and the data were -409.40 kcal/mol and 43.85%,-451.50 kcal/mol and 47.83%,-606.50 kcal/mol and 64.10%, and -384.70 kcal/mol and 38.01% for M16, Y16, P16 and W16, respectively. Y16 has a variation of five amino acids from M16, P16, and W16 (H202D, T266A, Δ424S, D441Δ, and K452Q), and interestingly Y16 showed a poor solubility and assembly into virus-like particle as compared to the others. Using M16 for further expression optimization, the results indicated that the deficiency of proteinase in engineered host strains facilitated the high expression of HPV16 L1; the use of plasmid vector pPink HC which theoretically guarantees high gene copy numbers in the selected colonies didn’t produce higher expression than the use of plasmid pPink LC; in addition, the recombinant proteins with N-terminal fusion of Glutathione S-transferase or alpha factor from Saccharomy cescerevisiae, or with C-terminal truncation failed to further improve expression level.Conclusions Major capsid L1 proteins from multi-genotypes of HPV were significantly expressed in soluble and GST-fusion forms in E. coli cells, which providing a valid alternative in terms of assembling and disassembling of VLPs in vitro. Furthermore, the results in the current study also added our knowledge to obtain high expression levels of heterologous genes in E. coli expression system. Besides, codon optimization is an applicable approach for improving expression of HPV16 L1 in P. pastoris, however, the actually effective mechanism might be complex since another important factor mRNA structure was also changed. In addition, protein stability in host cells, and variation in amino acid sequence have also significant influences in obtaining complete and properly-folded HPV16 L1 protein. Our results highlight that, the consequent changes of mRNA structure should be seriously considered while optimizing codon usage was commonly used as an efficient strategy to improve the expression of a heterogeneous gene; in addition, minor changes of amino acids sequence significantly reduced HPV L1 solubility and VLPs assembly.