Recombinant O -mannosylated protein production (PstS-1) from Mycobacterium tuberculosis in Pichia pastoris ( Komagataella phaffii ) as a tool to study tuberculosis infection

Background Pichia pastoris (syn. Komagataella phaffii ) is one of the most highly utilized eukaryotic expression systems for the production of heterologous glycoproteins, being able to perform both N - and O -mannosylation. In this study, we present the expression in P. pastoris of an O -mannosylated recombinant version of the 38 kDa glycolipoprotein PstS-1 from Mycobacterium tuberculosis ( Mtb ), that is similar in primary structure to the native secreted protein. Results The recombinant PstS-1 (rPstS-1) was produced without the native lipidation signal. Glycoprotein expression was under the control of the methanol-inducible promoter pAOX1, with secretion being directed by the α-mating factor secretion signal. Production of rPstS-1 was carried out in baffled shake flasks (BSFs) and controlled bioreactors. A production up to ~ 46 mg/L of the recombinant protein was achieved in both the BSFs and the bioreactors. The recombinant protein was recovered from the supernatant and purified in three steps, achieving a preparation with 98% electrophoretic purity. The primary and secondary structures of the recombinant protein were characterized, as well as its O -mannosylation pattern. Furthermore, a cross-reactivity analysis using serum antibodies from patients with active tuberculosis demonstrated recognition of the recombinant glycoprotein, indirectly indicating the similarity between the recombinant PstS-1 and the native protein from Mtb . Conclusions rPstS-1 (98.9% sequence identity, O -mannosylated, and without tags) was produced and secreted by P. pastoris , demonstrating that this yeast is a useful cell factory that could also be used to produce other glycosylated Mtb antigens. The rPstS-1 could be used as a tool for studying the role of this molecule during Mtb infection, and to develop and improve vaccines or kits based on the recombinant protein for serodiagnosis.