AUTHOR=Singhvi Priyank , Verma Juhi , Panwar Neha , Wani Tabiya Qayoom , Singh Akansha , Qudratullah Md. , Chakraborty Arnab , Saneja Ankit , Sarkar Debi P. , Panda Amulya K. 

TITLE=Molecular Attributes Associated With Refolding of Inclusion Body Proteins Using the Freeze–Thaw Method

JOURNAL=Frontiers in Microbiology

VOLUME=Volume 12 - 2021

YEAR=2021

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2021.618559

DOI=10.3389/fmicb.2021.618559

ISSN=1664-302X

ABSTRACT=Structure-function understanding of inclusion bodies (IBs) in the last two decades have led to the development of several mild solubilization buffers for improved recovery of bioactive proteins. Recently developed freeze-thaw based inclusion body proteins solubilization method has received a great deal of attention due to its simplicity and cost-effectiveness.  The present report investigates the reproducibility, efficiency and plausible mechanism of the freeze-thaw based IBs solubilization. Percentage recovery of functionally active protein species of human growth hormone (hGH) and L-asparaginase from their IBs in E. coli and the quality attributes associated with freeze-thaw based solubilization method was analyzed in detail. The overall yield of purified hGH and L-asparaginase protein was found to be around 14% and 25% respectively. Both the purified proteins had functionally active species lower than that observed with commercial proteins. Biophysical and biochemical analysis revealed that the formation of soluble aggregates was a major limitation in the case of tough IB protein like hGH. On the other hand, the destabilization of soft IB protein like L-asparaginase led to the poor recovery of functionally active protein species. Our study provides an insight into the advantages, disadvantages and molecular-structural information associated with freeze-thaw based solubilization method.