Downloads: 142 | Views: 197
Research Paper | Microbiology | India | Volume 5 Issue 10, October 2016
Synthesis of Purified Pneumolysin and Autolysin Proteins of S. pneumoniae in Prokaryotic System by Modified Recombinant DNA Technology
Feroze Ganaie | Geetha Nagraj | Vandana Govindan | Ravi Kumar KL
Abstract: This study aimed to produce purified recombinant proteins encoded by ply and lytA genes of S. pneumoniae in prokaryotic system. Gene specific primers were used to amplify ply and lytA genes from S. pneumoniae ATCC 49619 strain. The ply and lytA PCR products and pUC29 cloning vector were digested, ligated and transformed into E. coli (DH5). Sequence confirmed ply and lytA genes were sub cloned in pET28a expression vector. Proteins were over expressed by IPTG induction and purified using Ni2+-NTA based affinity column purification with imidazole gradient. PCR amplicons of size ~1413 bp for ply and ~954 bp for lytA were obtained. Sequencing analysis of clones revealed 100 % match with the ply and lytA genes of S. pneumoniae. Expression of proteins was maximum at 16 hours of IPTG induction. Higher quantity of ply and lytA proteins were found in 500mM imidazole elution with >90 % purity. Methods employed in prior studies for the production of ply and lytA proteins were laborious, time consuming and contributes poor yield and purity. Our strategy offers efficient and economical approach for the production of pure recombinant ply & lytA proteins.
Keywords: Streptococcus pneumoniae, Pneumolysin, Autolysin, Recombinant proteins, Ni2+-NTA affinity chromatography
Edition: Volume 5 Issue 10, October 2016,
Pages: 179 - 184