Improvement the expression and purification of Loa22: a lipoprotein with OmpA domain from pathogenic Leptospira serovars

Abstract

Outer membrane proteins (OMPs) are the main surface antigens of the pathogenic Leptospires. One of the highly conserved outer membrane proteins expressed only by pathogenic Leptospires is Loa22. This study aims is to obtain the optimum conditions for high expression and purification of Loa22 recombinant (rLoa22) protein. Based on evidence of phylogenetic studies, complete coding sequence of loa22 gene was optimized based on codon usage chart and sub-cloned into a pET32a (+) expression vector. BL21 (pLysS) was used as expression host for transformation. The recombinant clones selected on ampicillin plates and subjected to PCR by using pET T7 primers and expression conditions optimized then by adjusting parameters such as culture media, induction time, temperature, and IPTG concentration. SDS-PAGE Analysis showed that the production of rLoa22 protein was at the highest level when post induction incubation, IPTG concentration, and duration of induction were 37ºC, 0.1 M and 5 h in 2xTY medium respectively. Due to the soluble nature of the protein, the purification of the rLoa22 protein under native conditions using Ni-NTA pull-down was optimum in one hour binding process at 37°C, five times washing process and elution buffer with a pH 7.4 and a 0.3 M imidazole concentration. Based on the results of this study, optimizing the expression and purification process for over production of rLoa22 protein resulted in the large quantity of pure recombinant antigen that forms the basis for future investigation on the design of rapid diagnostic tests and more effective subunit vaccine candidates for leptospirosis.