Investigation of pH and nisin inducible-promoters in lactic acid bacteria for recombinant protein production
Loo, Bao Ren
Date of Issue2015
School of Biological Sciences
A*STAR Bioprocessing Technology Institute
Food-grade lactic acid bacteria such as Lactococcus lactis possess enormous commercial applications, and recombinant protein production has progressively been explored in these cells. However, currently only nisin inducible-expression system is commercially-available. This study aim to evaluate the use of alternative inducible-systems for recombinant protein production in L. lactis. Cells carrying eGFP genes under the control of nisin or pH inducible-promoters were cultivated to mid-log phase and induced as followed: PnisA with 10 ng ml-1 nisin (at pH 5 and 7); P170 and PgroESL with an acidic shift to pH 5 from pH 7. Western blot, fluorescence microscopy and quantitative PCR were performed. Our results showed successful expression of eGFP using PnisA. However, at pH 5, fold change of eGFP transcription level was significantly lower (from 197.0- to 48.0-fold), and no eGFP was detected using Western blot and fluorescence microscopy. Induction of P170 and PgroESL showed minimal fold change in eGFP transcription (1.9- and 2.1-fold, respectively), with no eGFP detected from Western blot and fluorescence microscopy. Proof-of-concept demonstration using PnisA with commercially important proteins, aspergillopepsin and cysteine endoprotease, succeeded with both proteins detected in Western blot. In conclusion, pH inducible-promoters showed poorer expression over the nisin-inducible PnisA system in our study.
Final Year Project (FYP)
Nanyang Technological University