CLONING AND EXPRESSION OF L1 HPV 52 GENE ISOLATED FROM INDONESIA IN Escherichia coli BL21(DE3)

• Main Author: Anjayati, Henik
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/42107
• Institution: Institut Teknologi Bandung

Cervical cancer is the third woman cancer in the world and is the first cancer cause in Indonesia. It is caused by a Human Papillomavirus (HPV) with a high-risk infection. The most common high-risk infection of HPV in Indonesia is HPV 52. This HPV infection can be prevented by a VLP-based vaccine assembled from the L1 HPV capsid protein. Thus, the VLP-based vaccine developed from Indonesian L1 HPV 52 is expected to be more effective in preventing HPV infection in Indonesia. This study purposed to clone the L1 HPV 52 gene isolated from Indonesia in pET32b(+) and to express the L1 HPV 52 capsid protein in E. coli BL21(DE3). The method used included in silico study which consisted of L1 HPV 52 ligation simulation on pET32b(+), molecular weight and pI analysis, hydrophobicity analysis, transmembrane analysis, and structure analysis using Snapgene 2.3.2, ExPASy, fasta.bioch.virginia.edu/, wlab.ethz.ch/protter/#, and i-Tasser respectively. The second step was to clone the recombinant plasmid pET32b(+)-L1 HPV 52 confirmed by using PCR, restriction and DNA sequencing. The next step was to express the recombinant L1 HPV 52 protein with IPTG induction and without IPTG induction. The recombinant protein expressed by IPTG induction was carried out induction duration and IPTG concentration optimization. The optimization results were continued by recombinant protein purification. All of the protein expression and protein purification results were analyzed by SDS PAGE and ImageJ. In silico studies showed that the L1 HPV 52 amplicon was 1448bp and would produce a recombinant protein of ~70.9kDa in size with pI 6.360, had a hydrophilic tendency and had no transmembrane region. PCR result was DNA of ~1500bp, restriction by using a single digest was DNA of ~8000bp, and sequencing result was in frame with native L1 HPV 52. IPTG induction result showed a thicker protein band of ~70.9kDa with the optimum conditions of 0.5mM IPTG, 370C for 5 hours, but recombinant protein L1 HPV 52 was an insoluble phase. Therefore, the optimum concentration of urea obtained from the optimization results was needed to dissolve the recombinant protein, which was 8M. The recombinant protein was then purified using NI-NTA agarose with urea variation for refolding buffer and imidazole variation for elution buffer. The purification results showed that almost the recombinant protein came out with flow through. It was possible that 6xHis•Tag wasn’t bound to the resin. From this study, it can be concluded that L1 HPV 52 gene is successfully cloned and expressed in E. coli Bl21(DE3), but it still needs the optimization of purification conditions to purify recombinant proteins.