Optimization of Inclusion Body Formation and Purification in Multi-well Plates

Abstract

Heterologous expression has long been used for the efficient production of proteins and enzymes as it offers significant advantages over purification of proteins from their native organisms. When first established, great efforts have been made to heterologously express proteins with high yields in the soluble fraction, hence, avoiding protein aggregation. In recent decades, however, it has been shown that the formation of aggregates (inclusion bodies; IBs) can be beneficial. To recover active protein, however, proteins should have been refolded from IBs after purification. The discovery that IBs themselves can also be active has revolutionized the entire protein production field. Therefore, several approaches have been described to generate catalytically active IBs during heterologous expression. Since several extrinsic and intrinsic factors such as protein structure and toxicity, pH and temperature of expression, and the used media might influence the formation of IBs, it is time and material consuming to use shake flask to examine and optimize different expression conditions. However, by using multi-well plates, it is possible to rapidly develop an efficient protocol for the expression of catalytically active IBs in a rational approach. The presented protocol was used for the heterologous expression of a 5'-adenosine monophosphate phosphorylase which forms catalytically active aggregates during expression in E. coli.

Keywords: Catalytically active IB; Expression conditions; Expression optimization; In-vivo formed IB; Parallel cultivation.