Characterisation of a novel stimuli-responsive polymers for protein refolding

• Main Author: Ang, Jing Mei
• Other Authors: Susanna Leong Su Jan
• Format: Final Year Project
• Language: English
• Published: 2009
• Subjects: DRNTU::Engineering::Chemical engineering::Biotechnology
• Online Access: http://hdl.handle.net/10356/16651
• Institution: Nanyang Technological University

During bioprocessing, there are many points at which protein can aggregate, reducing the overall protein refolding yield. A novel stimuli responsive polymer: Poly (L-lysine iso-phthalamide) was investigated in this study to examine its potential use as an additive to improve refolding yield of hen egg white lysozyme (HEWL). Poly (L-lysine iso-phthalamide) is anionic at pH 6.0 or higher and cationic when the environmental pH falls below pH 4.0. Phase transition of the polymer from soluble state to an insoluble state occurs between pH 4.0 to 6.0. Investigations were then carried out to determine the optimal conditions to carry out lysozyme refolding. Factors that influence lysozyme refolding yield such as (i) protein refolding pH, (ii) polymer concentration in refolding buffer and (iii) urea concentration in refolding buffer were examined. Maximum lysozyme refolding yield of 22% was achieved when refolding was carried out using a refolding buffer consisted of 0.2 W/V% Poly (L-lysine iso-phthalamide), 2.0M urea and 4mM EDTA at pH 8. A control study was also carried out to investigate the effects of GSH and GSSG on lysozyme refolding yield. GSH and GSSG are redox agents that are normally added into refolding buffer to improve protein refolding yield. The addition of GSH and GSSG helps improve lysozyme refolding yield as they promote disulfide bond formation. However, Poly (L-lysine iso-phthalamide) was found to be ineffective in improving lysozyme refolding yield when GSH and GSSG were added into the refolding buffer. In fact, 68% lysozyme refolding yield was achieved when no polymer was used in the presence of GSH and GSSG. When the polymer is added into this refolding buffer, lysozyme refolding yield was reduced to less than 10%. This finding suggests that possible molecular interactions between the polymer, GSH and GSSG are inhibiting lysozyme refolding hence the decrement in refolding yield.