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Current Trends in Biotechnology and Pharmacy Vol. 5 (3) 1309-1317 July 2011, ISSN 0973-8916 (Print), 2230-7303 (Online) 1313 5 after codon optimization (Fig. 1). Taken together the codon optimized gene sequence of HPV L2 was favourable for expression in Pichia pastoris. 3.1 Cloning and Integration of HPV16L2 gene into Pichia pastoris: Human papilloma virus 16 L2 gene codon optimized for expression in Pichia pastoris was cloned into expression vector pPICZB (Fig. 2). Cloning of the minor capsid protein 16 L2 gene into pPICZB was verified by restriction enzyme analysis using EcoRI and XhoI. A band of approximately 1.5 kb size was observed on agarose gel indicating the presence of L2 gene (Fig. 3A). The clone was confirmed by restriction analysis using XbaI which is located in 16L2 gene sequence as well as pPICZB vector backbone. The restriction digested plasmid revealed a band of approximately 800bp on agarose gel confirming the presence of HPV 16 L2 gene (Fig. 3B). Further, DNA sequencing of the PCR amplicons using Alcohol Oxidase (AOX) primers confirmed the identity of the integrated gene upon BLAST search (data not shown). HPV 16 L2 gene was integrated into Pichia pastoris genome by transforming the chemically competent Pichia pastoris cells using the recombinant plasmids. The integration was PCR verified using primers that either bind at AOX promoter site or HPV L2 gene. The PCR products showed the bands size of approximately 1.8 kb and 1.5kb with AOX primers and HPV 16 L2 gene specific primers respectively (Fig. 4A & 4B). 3.2 Reverse Transcriptase PCR: Recombinant Pichia pastoris clones expressing HPV16 L2 were induced for protein expression using 0.5% (v/v) methanol. An aliquot of cells were withdrawn after 24, 48 and 72 hours of induction. Total RNA were extracted from these induced Pichia pastoris, produced an amplification Percentage of Codons Codon Usage (frequency distribution) Fig. 1. Comparative Analysis of various parameters employed for codon optimization. The bar graph shows frequency distribution of codon usage. The native HPV 16L2 had 39% of codons that were most preferred in Pichia pastoris, while the optimized gene sequence had 72% codons that were most preferred in Pichia pastoris. The graph in the inset shows Codon adaptation index, Overall GC content and CIS negative elements present in the native and codon optimized HPV 16 L2 DNA sequences. All the parameters show a significant improvement over the native L2 DNA sequence. Fig. 2. Schematic diagram of HPV16 L2 constructs. The filled box represent HPV16 L2 gene. Unique restriction enzyme sites present within HPV 16L2 are marked with nucleotide position in parenthesis. HPV16 L2 gene was cloned downstream of AOX promoter between EcoRI and XhoI enzyme sites. Expression of recombinant minor capsid protein of HPV in Pichia pastoris
Current Trends in Biotechnology and Pharmacy Vol. 5 (3) 1309-1317 July 2011, ISSN 0973-8916 (Print), 2230-7303 (Online) 1314 gene amplification from possible DNA contamination in extracted RNA was ruled out in a PCR reaction that employed identical RNA samples isolated from induced Pichia pastoris cells. Fig. 3. Restriction enzyme analysis of HPV 16L2 cloned in pPICZB. The DNA fragments were resolved on 1% Agarose gel and visualized by Ethedium Bromide staining. A. HPV 16 L2 clone digested with EcoRI & XhoI Lane M-Gene ruler, 1Kbp ladder (Fermentas), Lane ‘16L2’ indicates E. coli clone with the released 16 L2 gene of approximately 1500 bp in size. B. HPV 16 L2 clone digested with restriction enzyme XbaI, 1Kbp ladder (Fermentas), Lane ‘16L2’ indicates clone showing the released 16 L2 gene of approximately 800 bp in size. Fig. 4. A. PCR amplification of HPV 16L2 clone using AOX promoter specific primers. The PCR products were resolved on 1% agarose gel. Lane 1 16 L2 Pichia clone, Lane 2 negative control, Lane 3 positive control (pPICZB 16L2 plasmid used for transforming the Pichia pastoris genome) and Lane M 1 Kbp ladder (Fermentas). The amplified bands corresponds approximately 1.8 Kbp size B. PCR amplification HPV 16 L2 region using Gene specific primers .The PCR products were resolved on 1% agarose gel. Lane 1 positive control (pPICZB 16L2 plasmid used for transforming the Pichia pastoris genome), Lane M 1Kbp ladder (Fermentas), Lane 2 HPV 16L2 Pichia clone and Lane 3 negative control. product of approximately 1.5kb as seen on agarose gel. RT-PCR analysis was performed for recombinant HPV16 L2 Pichia clone and the assay confirmed that the gene is transcribed on induction with methanol (Fig. 5). The HPV L2 Fig. 5. RT PCR analysis of methanol induced HPV 16 L2 Pichia pastoris clone: RT-PCR of methanol induced Pichia pastoris clone expressing HPV 16 minor capsid protein after 24 hr (lane 1), 48 hr (lane 2) & 72 hours (lane 3) post induction.; PCR (without reverse transcription reaction) of RNA samples obtained from induced Pichia pastoris clones expressing HPV 16 major capsid protein after 24 hr (lane 4), 48hr (lane 5) and 72 hours (lane 6). GS: Naïve Pichia pastoris strain GS115. Lane M 1Kbp ladder (Fermentas). The amplified 16 L2 product corresponds to the approximately 1.5kb in size. 3.3 Immuno Blotting : The cell lysates of recombinant Pichia pastoris were analyzed in Western blot using HPV L2 peptide specific mouse monoclonal antibody, which showed the staining of band size of approximately 55KDa. This experiment proved beyond a reasonable doubt, the expression of HPV16 L2 in Pichia pastoris. The mouse monoclonal antibody did not react with naïve Pichia cell lysate used as negative control (Fig.6). In the Western blot apart from the 55KDa band, which is the expected size of L2 a higher molecular weight protein was also observed. There are reports indicating the frequent association of Hsp 70 family proteins with HPV L2. The Hsp70 proteins are thought to act like chaperon that help protein fold or unfold (23). Some investigators observed that apart from HSPs the sumoylation proteins are also tightly associated with HPV L2 (24). The higher size protein band corresponding to HPV L2 observed in this study might be a consequence of the association of any of these chaperones. Further Hanumantha Rao et al
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(December 7-9, 2011) Venue: Karunya
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