N-Propanol Based Solubilization Buffer Enhances Refolding Yield of Inclusion Body Protein by Populating Intermediates to the Folding Pathway

Most of the times, high level expression of recombinant proteins in bacteria results in accumulation of recombinant proteins into inclusion body (IB) aggregates. To obtain native protein from these aggregates, it is necessary to solubilize these aggregates followed by refolding of solubilized protein by appropriate refolding method. Conventionally, high concentration of denaturant like urea or guanidinium chloride (GdmCl) is used for solubilization of inclusion bodies which often results into aggregation of protein during refolding process. In the present study we have evaluated a novel solubilization method using n-propanol in presence of low concentration of urea. n-Propanol based solubilization agent was compared with traditional solubilization agents like 8 M urea and 6 M GdmCl for solubilization efficiency, structure and stability of the solubilized model protein, recombinant human growth hormone (hGH). hGH IBs were found to be tough and were only solubilized efficiently in presence of high concentration of denaturants (8 M urea or 6 M GdmCl). 4 to 6 M n-propanol in combination with 2 M urea was sufficient for the efficient solubilization of hGH IBs. Aggregation during refolding was also studied and it was found that solubilization with n-propanol based buffer resulted into bioactive hGH without aggregation giving significantly higher refolding yield in comparison to those obtained with urea and GdmCl based buffers which resulted in aggregation of hGH during refolding. From the results obtained, it can be concluded that solubilization of hGH IBs in n-propanol based buffer results in a partially folded folding intermediate of hGH which readily folds into native form on dilution with reduced chances of protein getting into aggregation pathway.