Co-expression of protein chaperones for recombinant protein production in lactic acid bacteria

• Main Author: Tay, Stephanie Victoria Choo Gek
• Other Authors: Dave Ow Siak Wei
• Format: Final Year Project
• Language: English
• Published: 2016
• Subjects: DRNTU::Science::Biological sciences
• Online Access: http://hdl.handle.net/10356/66963
• Institution: Nanyang Technological University

Lactococcus lactis is a promising microbial cell factory for the production of recombinant proteins like Nattokinase (NK), a key bio-therapeutic enzyme for cardiovascular diseases. During expression, cells could exhibit stress response due to the presence of protein aggregates. Currently, limited studies are conducted on co-expressing molecular chaperones to refold protein aggregates in L. lactis. This study aims to investigate whether constitutive co-expression of chaperones facilitate protein folding of NK in L. lactis. Western blot and fibrin assay demonstrated that pNZ8148-NK and pNZ8148-USP45-NK expressed from 100 ng/mL over 10 ng/mL of nisin had higher expression and activity in soluble and secreted fractions while insoluble fractions had no functional activity. Attempts at cloning of constitutive promoters: p59 and pUSP45 and chaperones: ClpX, Dnak, Hsp33 and Trigger Factor from Lactobacillus plantarum using T4 ligase cloning and Gibson assembly led to the successful cloning of constitutive promoters into pORI198 co-expression plasmid but not in pNZ9530. Further cloning optimization is required to clone the chaperones into pORI198 and introduce into pNZ8148-NK-NZ9000 and pNZ8148-USP45-NK-NZ9000 cells to improve NK protein folding. We hope to gain insights on the role of molecular chaperones in facilitating protein folding and improve L. lactis as an expression system for recombinant protein production.