Expression, purification, and characterization of self-assembly virus-like particles of capsid protein L1 HPV 52 in Pichia pastoris GS115
Chindy Nur Rosmeita, Sri Budiarti, Apon Zaenal Mustopa, Ela Novianti, Sri Swasthikawati, Sheila Chairunnisa, Ai Hertati, Maritsa Nurfatwa, Nurlaili Ekawati, Nurhasni Hasan
Applications: ELISA
Reactive species: HPV52
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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."
Article snippet:
The HPV 52 L1 protein was examined by ELISA using specific antibodies (Creative Diagnostics, USA).
Figure 1. Western blot analysis of the HPV 52 L1 protein in the fractions collected from the cation exchange column.