High yield expression of leptospirosis vaccine candidates LigA and LipL32 in the methylotrophic yeast Pichia pastoris

Abstract

BackgroundLeptospirosis, a zoonosis caused by Leptospira spp., is recognized as an emergent infectious disease. Due to the lack of adequate diagnostic tools, vaccines are an attractive intervention strategy. Recombinant proteins produced in Escherichia coli have demonstrated promising results, albeit with variable efficacy. Pichia pastoris is an alternative host with several advantages for the production of recombinant proteins.ResultsThe vaccine candidates LigANI and LipL32 were cloned and expressed in P. pastoris as secreted proteins. Large-scale expression resulted in a yield of 276 mg/L for LigANI and 285 mg/L for LipL32. The recombinant proteins were glycosylated and were recognized by antibodies present in the sera of patients with severe leptospirosis.ConclusionsThe expression of LigANI and LipL32 in P. pastoris resulted in a significant increase in yield compared to expression in E. coli. In addition, the proteins were secreted, allowing for easy purification, and retained the antigenic characteristics of the native proteins, demonstrating their potential application as subunit vaccine candidates.

Recombinant LipL32 Protein Developed Using a Synthetic Gene Detects Leptospiraspecific Antibodies in Human Serum Samples

“…The rLipL32 was expressed with a His-Tag at the C-terminus in E. coli under the control of an IPTG-induced T7 promoter. SDS-PAGE analysis showed that the rLipL32 expressed was approximately 40 kDa in size, which differs from what has been reported in the literature (9,19,20,21,22,23). The synthetic lipl32 gene in this study was designed without its own start codon (ATG), and the expression of rLipL32 was initiated by the start codon from the pET22b expression vector, thus causing an increase in the molecular weight of the rLipL32 protein.…”

Expressão heteróloga da EMA-2 (equi merozoite antigen) de Theileria equi em Pichia pastoris com potencial utilização em imunobiológicos

“…O sistema de expressão P. pastoris que foi utilizado para a expressão da proteína EMA-2 de T. equi, proporciona um aumento na produção da proteína, se comparada ao sistema E. coli, pois as proteínas são secretadas para o meio facilitando sua purifi cação (HARTWIG et al, 2010).…”

Production of leptospiral LipL32 antigen in Pichia pastoris and its use in an enzyme-linked immunosorbent assay

“…These results demonstrated that ELISA developed here could be used as a screening test before confirmation by the MAT. The large-scale production of proteins with potential diagnostic value is important for the pharmaceutical, biomedical and biotechnological applications (Cos et al 2006;Hartwig et al 2010). An efficient secretion system is of great interest since these proteins may be easily recovered from the culture supernatant and maintain the conformational structure (Hartwig et al 2010).…”

“…The expression of rLipL32 MutS secretory phenotype was performed using the eukaryotic system based in P. pastoris, as described previously (Hartwig et al 2010). Briefly, a recombinant clone was grown in a baffled flask containing BMGY broth (1% yeast extract, 2% peptone, 1.34% yeast nitrogen base, 0.00004% biotin, 1 % glycerol, 100 mM potassium phosphate and 2 % agar, pH 6.0).…”

“…The production of recombinant LipL32 protein (rLipL32) in Escherichia coli has been extensively used for the development of vaccines (Adler and Moctezuma, 2010) and diagnostic tests for human (Flannery et al 2001), cattle (Bomfim et al 2005), dog (Dey et al 2004) and swine (Hartleben et al 2012). However, this system has demonstrated potential limitations, including low yield (Hartwig et al 2010), improper folding and lack of post-translational modifications (Cos et al 2006). Pichia pastoris has emerged as an important alternative expression system, because this yeast has the ability of growing in minimal medium at very high cell densities, secreting the heterologous protein simplifying its recovery (Cos et al 2006), and perform many of the post-translational modifications such as processing of signal sequences, folding, disulfide bridge formation, certain types of lipid addition, and O and N-linked glycosylation Cregg et al 2000;Hohenblum et al 2004;Cos et al 2006).…”