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Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris
Published on: February 25, 2010
Functional evaluation of a recombinant fungal immunomodulatory protein from L. rhinocerus produced in P. pastoris and
Udochukwu Camillius Ejike1, Chong Joo Chan1, Crystale Siew Ying Lim1
1Department of Biotechnology, Faculty of Applied Sciences, UCSI University, No.1, Jalan Menara Gading, UCSI Heights, 56000, Cheras, Wilayah Persekutuan Kuala Lumpur, Malaysia.
Abstract:
Fungal immunomodulatory proteins (FIPs) are bioactive proteins with immunomodulatory properties. We previously reported the heterologous production in Escherichia coli of FIP-Lrh from Tiger milk mushroom (Lignosus rhinocerus) with potent cytotoxic effect on cancer cell lines. However, protein produced in E. coli lacks post-translational modifications and may be contaminated with lipopolysaccharide (LPS) endotoxin. Therefore, in this study, yFIP-Lrh produced in Pichia pastoris was functionally compared with eFIP-Lrh produced in E. coli. Expression construct of FIP-Lrh cDNA in pPICZα was generated, transformed into P. pastoris X-33 and Mut+ transformants were verified by colony PCR. Induction with 0.5% or 1% methanol resulted in a secreted 13.6 kDa yFIP-Lrh which was subsequently purified and verified using LCMS/MS analysis. Size exclusion chromatography confirmed eFIP-Lrh as a homodimer whereas the larger size of yFIP-Lrh may indicate post-translational modification despite negative for glycoproteins staining. At lower concentration (4-8 μg/mL), yFIP-Lrh induced significantly higher Th1 (IFN-γ, TNF-α) and Th2 (IL-6, IL-4, IL-5, IL-13) cytokines production in mice splenocytes, whereas 16 μg/mL eFIP-Lrh induced significantly higher pro-inflammatory cytokines (TNF-α, IL-6, IL-10), possibly due to higher residual LPS endotoxin (0.082 EU/mL) in eFIP-Lrh compared to negligible level in yFIP-Lrh (0.001 EU/mL). Furthermore, yFIP-Lrh showed higher cytotoxic effect on MCF-7 and HeLa cancer cells. Since both recombinant proteins of FIP-Lrh have the same peptide sequence, besides glycosylation, other post-translational modifications in yFIP-Lrh may account for its enhanced immunomodulatory and anti-proliferative activities. In conclusion, P. pastoris is preferred over E. coli for production of a functionally active yFIP-Lrh devoid of endotoxin contamination. KEY POINTS: • FIP-Lrh can induced production of Th1 and Th2 cytokines by mouse splenocytes. • Higher cytotoxic effect on cancer cells observed for yeast compared to E. coli produced FIP-Lrh. • P. pastoris allows production of an endotoxin-free and functionally active recombinant FIP-Lrh.
Insights
Fungal immunomodulatory proteins (FIPs) like FIP-Lrh are better produced in Pichia pastoris for enhanced activity. Yeast-produced FIP-Lrh shows superior immunomodulatory and cytotoxic effects compared to E. coli produced protein, free from endotoxin contamination.
Area of Science:
- Biochemistry
- Immunology
- Biotechnology
Background:
- Fungal immunomodulatory proteins (FIPs) possess bioactive properties.
- Previous production of FIP-Lrh in E. coli yielded cytotoxic effects but lacked modifications and contained endotoxin.
- Recombinant protein production systems impact protein function and purity.
Purpose of the Study:
- To compare the functional activity of FIP-Lrh produced in Pichia pastoris (yFIP-Lrh) versus E. coli (eFIP-Lrh).
- To assess the immunomodulatory and cytotoxic effects of both recombinant proteins.
- To evaluate the impact of post-translational modifications and endotoxin levels on protein activity.
Main Methods:
- FIP-Lrh cDNA was expressed in Pichia pastoris X-33.
- Recombinant proteins were purified and analyzed using LC-MS/MS and size exclusion chromatography.
- Immunomodulatory effects were tested on mice splenocytes measuring cytokine production (Th1/Th2).
- Cytotoxic effects were evaluated on MCF-7 and HeLa cancer cell lines.
- Endotoxin levels were quantified.
Main Results:
- P. pastoris produced a 13.6 kDa secreted yFIP-Lrh, potentially with post-translational modifications.
- yFIP-Lrh induced significantly higher Th1 and Th2 cytokine production at lower concentrations compared to eFIP-Lrh.
- eFIP-Lrh showed higher pro-inflammatory cytokine induction at higher concentrations, attributed to higher residual LPS endotoxin.
- yFIP-Lrh exhibited a greater cytotoxic effect on cancer cell lines.
- yFIP-Lrh had negligible endotoxin levels (0.001 EU/mL) compared to eFIP-Lrh (0.082 EU/mL).
Conclusions:
- Pichia pastoris is a preferred host for producing endotoxin-free and functionally active FIP-Lrh.
- Post-translational modifications in yFIP-Lrh likely enhance its immunomodulatory and anti-proliferative activities.
- Recombinant FIP-Lrh production in P. pastoris offers advantages over E. coli for therapeutic applications.
