The effect of cell disruption techniques and chaotropic agents on the downstream purification process of mecasermin produced as inclusion body in E. coli

Abstract

The isolation of the target protein from inclusion bodies (IBs) is a preliminary step to increase protein titer and to maintain its biological activity. In the present study, the effects of various cell lysis methods and the expression temperature was investigated on the improvement of the subsequent purification steps of mecasermin produced in IB. We also investigated the solubilization profile of the top-notch IB in 6 M guanidine hydrochloride (Gdn-HCl) and 8 M urea at different pH ranges. Mecasermin was expressed at various temperatures (25, 28, 30, and 37 °C) and the Escherichia coli cells were lysed by different cell lysis methods. The purity and quality of harvested IBs was evaluated with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Finally, mecasermin was refolded and purified using gel filtration chromatography. The profile of SDS-PAGE analysis showed higher quality and purity after application of sonication coupled with lysozyme pretreatment for expressed mecasermin at 37 °C. Besides, from dithiothreitol application in washing step, we achieved a manifold enriched secondary IB for further purification of mecasermin. Mecasermin exhibited optimized solubility in 6 M Gdn-HCl at pH of 5.4. The findings of this study indicate an important role for cell disruption techniques to efficient purification of mecasermin. The study presents the most efficient techniques for improvement of downstream purification of mecasermin.

Keywords: Cell disruption; Inclusion body purification; Mecasermin; Strong chaotrope.

Fig. 1

The SDS-PAGE pattern of optimum cell lysis procedure (lysozyme pretreatment plus sonication) for IB formed mecasermin on 17.5% acrylamide gel. Different lanes contain the following: Lane 1; molecular weight marker (kDa), Lane 2; the lysate after sonication with lysozyme treatment in lysis buffer A, lane 3; first resulted light phase from washing with lysis buffer A. lane 4; first resulted sediment from washing with lysis buffer A, lane 5; the supernatant of second washing with lysis buffer A. lane 6; resulted sediment from secondary wash with buffer A, lane 7; resulted sediment from washing with buffer B (1 M urea + buffer A), lane 8; the supernatant of washing with buffer B, lane 9; final obtained washed pellet from washing with triton x-114 in buffer B. The position of mecasermin is indicated by the arrow.

Fig. 2

Documentation of SDS-PAGE analysis for mecasermin distributed between pellets and supernatant fractions after bacterial cell lysis by lysozyme treatment on 17.5%.gel. Different lanes contain the followings. Lane 1; the lysate after lysozyme treatment plus RNase and DNase. Lane 2; molecular weight marker (kDa), lane 3; the first pellet after washing with buffer A, lane 4; the light phase from first washing with buffer A, lane 5; resulted sediment from second washing with buffer A, lane 6; the supernatant of second wash with lysis buffer A, lane 7; resulted pellet from washing with buffer B (1m urea + buffer A); lane 8; the supernatant of first washing with lysis buffer B, lane 9; the resulted pellet obtained from second washing with buffer B along with triton x-114, Lane 10; the final resulted supernatant of final washing with triton x-114 in buffer B. The position of IGF-I and lysozyme is indicated by the arrows.

Fig. 3

Western blotting panel for final washed pellet from washing phase A; in the presence of 5 mM DTT or B; absence of DTT. In A: lane 1 contains western blotting multicolor ladder and lane 2 contains the final pellet of washing process with buffer B containing DTT. In B: lane 1 contains western blotting multicolor ladder and lane 2 contains the final washed pellet of washing process with buffer B without DTT. In the B the bands at positions of 15 kDa and 24 kDa, are indicative of the presence of IGF-I antibody against mecasermin polypeptides aggregates as ligands.

Fig. 4

The SDS-PAGE analysis of eluted fractions of the gel filtration column with Superdex beads. Lane 1; molecular weight marker. Lane 2, 3; eluted fractions containing protein impurities with molecular weight greater than mecasermin. Lane 4; eluted fractions containing pure mecasermin.