Enhancement in the production of recombinant human paraoxonase 1 in Escherichia coli: A comprehensive approach of cellular engineering and optimization of protein folding process in vitro

Abstract

Human paraoxonase 1(hPON1) belongs to the paraoxonase (PON) family. It is a calcium-dependent enzyme with a size of ∼43 kDa and is composed of 6 bladed beta-barrel structures with two calcium ions in its active site. In humans, it is synthesized in the liver and remains bound with the high-density lipoproteins (HDL) within the blood. It has immense potential to tackle the poisoning associated with the use of organophosphates (OPs) and their derivatives, such as nerve agents, due to role in their degradation. Therefore, hPON1 serves as a potential bio-scavenger that can be used as an antidote or as a surface decontaminating agent in OPs poisoning. However, present systems prove insufficient to produce it in sufficient quantity to make it industrially relevant. Here, our efforts involve producing it recombinantly in an E. coli system with enhanced expression levels by altering cellular and environmental conditions. This has been further improved by the development of in-vitro refolding process for the denatured recombinant hPON1 (rhPON1) protein. This methodology resulted in approximately 200 mg of the enzymatically functional protein from 1 l of E. coli culture. Proper refolding of rhPON1 was confirmed by comparing its enzymatic activity and conformation with serum purified hPON1.