Abstract
BackgroundPichia 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.ResultsThe 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.ConclusionsrPstS-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.
Highlights
The selected clones were cultured in a 50 mL BMGY culture which was used to inoculate a 250 mL culture in baffled shake flasks (BSFs), and after 32 h in BMMY media and following the addition of 5, 10, or 20 mL/L of methanol, the growth and production of recombinant PstS-1 (rPstS-1) were compared
The data obtained in this study show peaks that are similar to those in the circular dichroism (CD) spectrum obtained from the recombinant 38 KDa reference antigen from the WHO bank, which has negative peaks at 208 nm and 225 nm [12], and the spectrum from the full length rPstS-1 expressed in E. coli which shows negative peaks at 208 nm and 218 nm, in addition to a positive peak at 190 nm [92]
The data obtained in this study suggest that the secondary structure of rPstS-1 produced in P. pastoris differs from that produced in bacteria
Summary
It was estimated that approximately 600,000 people developed multidrug-resistance (MDR) to TB [1] These data reflect the urgency in understanding the pathogenicity of Mtb, as well as the necessity to develop new therapeutic alternatives to treat or prevent the disease. The use of recombinant antigens to detect both latent and active TB provides for a simpler and more accurate diagnosis compared to other diagnostic tests in use today, such as the purified protein derivative (PPD) skin test or the Q uantiFERON®-TB test [18,19,20,21] For these reasons, it is important to be able to produce antigens with conformations that are as similar as possible to the native antigens produced by the mycobacteria so they can used as reagents in the development of diagnostic tools or vaccines
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