Abstract
A tetrameric protein of therapeutic importance, Escherichia coli L-asparaginase-II was expressed in Escherichia coli as inclusion bodies (IBs). Asparaginase IBs were solubilized using low concentration of urea and refolded into active tetrameric protein using pulsatile dilution method. Refolded asparaginase was purified in two steps by ion-exchange and gel filtration chromatographic techniques. The recovery of bioactive asparaginase from IBs was around 50%. The melting temperature (Tm) of the purified asparaginase was found to be 64°C. The specific activity of refolded, purified asparaginase was found to be comparable to the commercial asparaginase (190 IU/mg). Enzymatic activity of the refolded asparaginase was high even at four molar urea solutions, where the IB aggregates are completely solubilized. From the comparison of chemical denaturation data and activity at different concentrations of guanidine hydrochloride, it was observed that dissociation of monomeric units precedes the complete loss of helical secondary structures. Protection of the existing native-like protein structure during solubilization of IB aggregates with 4 M urea improved the propensity of monomer units to form oligomeric structure. Our mild solubilization technique retaining native-like structures, improved recovery of asparaginase in bioactive tetrameric form.
Highlights
Most of the times, expression of recombinant proteins in Escherichia coli leads to the formation of insoluble aggregates known as inclusion bodies (IBs) (Hartley and Kane, 1988; Fahnert et al, 2004)
We lysed the cells by sonication in the presence of lysozyme and IB pellet was extensively washed with 0.1% Na deoxycholate
Purified IB pellet was observed to consist of two major bands on SDS-PAGE gel, one corresponds to recombinant L-asparaginase (85%) and another one to the lysozyme used during IBs isolation (Figure 1B)
Summary
Expression of recombinant proteins in Escherichia coli leads to the formation of insoluble aggregates known as IBs (Hartley and Kane, 1988; Fahnert et al, 2004). Chaotropes such as urea and GdmCl completely denature the proteins and increases its propensity to aggregate during refolding resulting in low recovery of bioactive protein from IBs. IB proteins are reported to have structure and functional activities (Umetsu et al, 2004; Ventura and Villaverde, 2006; Peternel and Komel, 2011). Mild solubilization of IB aggregates protects the existing native-like protein structure of IB proteins and helps in its improved recovery into bioactive form Singh and Panda (2005). Solubilization with mild denaturing conditions has been proved to be more efficient for the recovery of bioactive protein from the IBs (Panda, 2003; Singh et al, 2012; Upadhyay et al, 2012)
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