Expression, purification, and characterization of self-assembly virus-like particles of capsid protein L1 HPV 52 in Pichia pastoris GS115

Abstract

Background: Cervical cancer caused by the human papillomavirus (HPV) is one of the most frequent malignances globally. HPV 52 is a high-risk cancer-causing genotype that has been identified as the most prevalent type in Indonesia. Virus-like particles (VLP)-based vaccinations against HPV infection could benefit from self-assembled VLP of L1 capsid protein.

Result: The recombinant HPV 52 L1 was expressed in Pichia pastoris on a shake-flask scale with 0.5% methanol induction in this study. The copy number was used to compare the expression level and stability. The colony that survived on a solid medium containing 2000 μg/ml of Zeocin was selected and cultured to express HPV 52 L1. DNA was extracted from the chosen colony, and the copy was determined using qPCR. HPV 52 L1 protein was then purified through fast performance liquid chromatography. Transmission electron microscopy (TEM) evaluation confirmed the VLP self-assembly. The genomic DNA remained intact after 100 generations of serial cultivation under no selective pressure medium conditions, and the protein produced was relatively stable. However, the band intensity was slightly lower than in the parental colony. In terms of copy number, a low copy transformant resulted in low expression but produced a highly stable recombinant clone. Eventually, the L1 protein expressed in Pichia pastoris can self-assemble into VLP. Therefore, recombinant HPV possesses a stable clone and the ability to self-assemble into VLP.

Conclusion: The recombinant L1 HPV 52 protein is successfully expressed in P. pastoris within a size range of approximately 55 kDa and demonstrated favorable stability. The L1 protein expressed in Pichia pastoris successful self-assembled of HPV VLPs, thereby establishing their potential efficacy as a prophylactic vaccine.

Keywords: Capsid protein L1; HPV 52; Protein purification; Recombinant Pichia pastoris; VLP-based vaccine.

Fig. 1

Western blot analysis of HPV 52 L1 expression in Pichia pastoris induced with 0.5% methanol at 48, 72, and 96 h of survived colonies on 2000 μg/ml Zeocin

Fig. 2

Stability analysis of the recombinant clone P. pastoris. A Genome DNA of 100 generations from selected clones showed persistent bands at 1500 bp, indicating a genetically stable clone. B Relative L1 protein intensity expressed from 100 generations indicated relative protein expression stability

Fig. 3

Purification of HPV 52 L1 by cation exchange. A Cation exchange chromatogram, B SDS-PAGE analysis, and C Western blot analysis of the HPV 52 L1 protein in the fractions collected from the cation exchange column. Lane M, molecular weight markers; lane C+, control of HPV 52 L1 designed by Creative Diagnostics (USA); lane FT, flowthrough; and lanes F1–F9, fractions from the elution peak. The arrows on the right in B and C indicate the position of HPV 52 L1

Fig. 4

Purification of HPV 52 L1 by size exclusion. A Size-exclusion chromatogram. B SDS-PAGE analysis of the HPV 52 L1 protein in the fractions collected from the size-exclusion column. Lane M, molecular weight markers; lane C+, control of HPV 52 L1 that was designed by Creative Diagnostics (USA); lanes F1–F5, fractions from the elution peak. The arrows on the right in B indicate the position of HPV 52 L1

Fig. 5

TEM analysis of L1 proteins. VLP is pointed by red arrow with a reference bar in the right-down corner of the box