full issue - Association of Biotechnology and Pharmacy
full issue - Association of Biotechnology and Pharmacy
full issue - Association of Biotechnology and Pharmacy
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Current Trends in <strong>Biotechnology</strong> <strong>and</strong> <strong>Pharmacy</strong><br />
Vol. 5 (3) 1309-1317 July 2011, ISSN 0973-8916 (Print), 2230-7303 (Online)<br />
1314<br />
gene amplification from possible DNA<br />
contamination in extracted RNA was ruled out<br />
in a PCR reaction that employed identical RNA<br />
samples isolated from induced Pichia pastoris<br />
cells.<br />
Fig. 3. Restriction enzyme analysis <strong>of</strong> HPV 16L2 cloned<br />
in pPICZB. The DNA fragments were resolved on 1%<br />
Agarose gel <strong>and</strong> visualized by Ethedium Bromide staining.<br />
A. HPV 16 L2 clone digested with EcoRI & XhoI Lane<br />
M-Gene ruler, 1Kbp ladder (Fermentas), Lane ‘16L2’<br />
indicates E. coli clone with the released 16 L2 gene <strong>of</strong><br />
approximately 1500 bp in size.<br />
B. HPV 16 L2 clone digested with restriction enzyme<br />
XbaI, 1Kbp ladder (Fermentas), Lane ‘16L2’ indicates<br />
clone showing the released 16 L2 gene <strong>of</strong> approximately<br />
800 bp in size.<br />
Fig. 4. A. PCR amplification <strong>of</strong> HPV 16L2 clone using<br />
AOX promoter specific primers. The PCR products were<br />
resolved on 1% agarose gel. Lane 1 16 L2 Pichia clone,<br />
Lane 2 negative control, Lane 3 positive control (pPICZB<br />
16L2 plasmid used for transforming the Pichia pastoris<br />
genome) <strong>and</strong> Lane M 1 Kbp ladder (Fermentas). The<br />
amplified b<strong>and</strong>s corresponds approximately 1.8 Kbp size<br />
B. PCR amplification HPV 16 L2 region using Gene<br />
specific primers .The PCR products were resolved on 1%<br />
agarose gel. Lane 1 positive control (pPICZB 16L2<br />
plasmid used for transforming the Pichia pastoris<br />
genome), Lane M 1Kbp ladder (Fermentas), Lane 2 HPV<br />
16L2 Pichia clone <strong>and</strong> Lane 3 negative control.<br />
product <strong>of</strong> approximately 1.5kb as seen on<br />
agarose gel. RT-PCR analysis was performed for<br />
recombinant HPV16 L2 Pichia clone <strong>and</strong> the<br />
assay confirmed that the gene is transcribed on<br />
induction with methanol (Fig. 5). The HPV L2<br />
Fig. 5. RT PCR analysis <strong>of</strong> methanol induced HPV 16 L2<br />
Pichia pastoris clone: RT-PCR <strong>of</strong> methanol induced Pichia<br />
pastoris clone expressing HPV 16 minor capsid protein<br />
after 24 hr (lane 1), 48 hr (lane 2) & 72 hours (lane 3) post<br />
induction.; PCR (without reverse transcription reaction)<br />
<strong>of</strong> RNA samples obtained from induced Pichia pastoris<br />
clones expressing HPV 16 major capsid protein after 24<br />
hr (lane 4), 48hr (lane 5) <strong>and</strong> 72 hours (lane 6). GS: Naïve<br />
Pichia pastoris strain GS115. Lane M 1Kbp ladder<br />
(Fermentas). The amplified 16 L2 product corresponds to<br />
the approximately 1.5kb in size.<br />
3.3 Immuno Blotting : The cell lysates <strong>of</strong><br />
recombinant Pichia pastoris were analyzed in<br />
Western blot using HPV L2 peptide specific<br />
mouse monoclonal antibody, which showed the<br />
staining <strong>of</strong> b<strong>and</strong> size <strong>of</strong> approximately 55KDa.<br />
This experiment proved beyond a reasonable<br />
doubt, the expression <strong>of</strong> HPV16 L2 in Pichia<br />
pastoris. The mouse monoclonal antibody did<br />
not react with naïve Pichia cell lysate used as<br />
negative control (Fig.6).<br />
In the Western blot apart from the 55KDa<br />
b<strong>and</strong>, which is the expected size <strong>of</strong> L2 a higher<br />
molecular weight protein was also observed.<br />
There are reports indicating the frequent<br />
association <strong>of</strong> Hsp 70 family proteins with HPV<br />
L2. The Hsp70 proteins are thought to act like<br />
chaperon that help protein fold or unfold (23).<br />
Some investigators observed that apart from<br />
HSPs the sumoylation proteins are also tightly<br />
associated with HPV L2 (24). The higher size<br />
protein b<strong>and</strong> corresponding to HPV L2 observed<br />
in this study might be a consequence <strong>of</strong> the<br />
association <strong>of</strong> any <strong>of</strong> these chaperones. Further<br />
Hanumantha Rao et al