Expression and purification of biologically active recombinant human paraoxonase 1 from inclusion bodies of Escherichia coli

• Published in: Protein expression and purification 2015-11, Vol.115, p.95-101
• Main Authors: Bajaj, Priyanka, Tripathy, Rajan K., Aggarwal, Geetika, Pande, Abhay H.
• Format: Article
• Language: English
• Subjects: Acyl homoserine lactone; Aryldialkylphosphatase - genetics; Aryldialkylphosphatase - isolation & purification; Aryldialkylphosphatase - metabolism; Base Sequence; Escherichia coli - genetics; Homocysteine thiolactone; Humans; In vitro refolding; Inclusion bodies; Inclusion Bodies - chemistry; Inclusion Bodies - metabolism; Molecular Sequence Data; Organophosphate; Protein Refolding; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2015.05.011

•Human paraoxonase 1 is a potential candidate for the therapeutic intervention in humans•Generation of recombinant h-PON1 (rh-PON1) in E. coli has been elusive till now•Our results suggest that rh-PON1 can be produced by in vitro refolding of inclusion bodies•Our method can be developed for large scale production of rh-PON1 enzymes•This will aid in developing h-PON1 as a therapeutic candidate Human PON1 (h-PON1) is a Ca2+-dependent serum enzyme and can hydrolyze (and inactivate) a wide range of substrates. It is a multifaceted enzyme and exhibit anti-inflammatory, anti-oxidative, anti-atherogenic, anti-diabetic, anti-microbial, and organophosphate (OP)-detoxifying properties. Thus, h-PON1 is a strong candidate for the development of therapeutic intervention against these conditions in humans. Insufficient hydrolyzing activity of native h-PON1 against desirable substrate affirms the urgent need to develop improved variant(s) of h-PON1 having enhanced activity. Production of recombinant h-PON1 (rh-PON1) using an Escherichia coli expression system is a key to develop such variant(s). However, generation of rh-PON1 using E. coli expression system has been elusive until now because of the aggregation of over-expressed rh-PON1 protein in inactive form as inclusion bodies (IBs) in the bacterial cells. In this study, we have over-expressed rh-PON1(wt) and rh-PON1(H115W;R192K) proteins as IBs in E. coli, and refolded the inactive enzymes present in the IBs to their active form using in vitro refolding. The active enzymes were isolated from the refolding mixture by ion-exchange chromatography. The catalytic properties of the refolded enzymes were similar to their soluble counterparts. Our results show that the pure and the active variant of rh-PON1 enzyme having enhanced hydrolyzing activity can be produced in large quantities using E. coli expression system. This method can be used for the industrial scale production of rh-PON1 enzymes and will aid in developing h-PON1 as a therapeutic candidate.