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Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models
Published on: November 11, 2014
Murine Factor H Co-Produced in Yeast With Protein Disulfide Isomerase Ameliorated C3 Dysregulation in Factor
Heather Kerr1,2, Andrew P Herbert2, Elisavet Makou2
1Centre for Inflammation Research, Queen's Medical Research Institute, University of Edinburgh, Edinburgh, United Kingdom.
Abstract:
Recombinant human factor H (hFH) has potential for treating diseases linked to aberrant complement regulation including C3 glomerulopathy (C3G) and dry age-related macular degeneration. Murine FH (mFH), produced in the same host, is useful for pre-clinical investigations in mouse models of disease. An abundance of FH in plasma suggests high doses, and hence microbial production, will be needed. Previously, Pichia pastoris produced useful but modest quantities of hFH. Herein, a similar strategy yielded miniscule quantities of mFH. Since FH has 40 disulfide bonds, we created a P. pastoris strain containing a methanol-inducible codon-modified gene for protein-disulfide isomerase (PDI) and transformed this with codon-modified DNA encoding mFH under the same promoter. What had been barely detectable yields of mFH became multiple 10s of mg/L. Our PDI-overexpressing strain also boosted hFH overproduction, by about tenfold. These enhancements exceeded PDI-related production gains reported for other proteins, all of which contain fewer disulfide-stabilized domains. We optimized fermentation conditions, purified recombinant mFH, enzymatically trimmed down its (non-human) N-glycans, characterised its functions in vitro and administered it to mice. In FH-knockout mice, our de-glycosylated recombinant mFH had a shorter half-life and induced more anti-mFH antibodies than mouse serum-derived, natively glycosylated, mFH. Even sequential daily injections of recombinant mFH failed to restore wild-type levels of FH and C3 in mouse plasma beyond 24 hours after the first injection. Nevertheless, mFH functionality appeared to persist in the glomerular basement membrane because C3-fragment deposition here, a hallmark of C3G, remained significantly reduced throughout and beyond the ten-day dosing regimen.
Insights
This study developed a novel Pichia pastoris strain to significantly increase recombinant murine factor H (mFH) production. While de-glycosylated mFH showed reduced half-life in mice, it effectively reduced C3 deposition in C3 glomerulopathy models.
Area of Science:
- Biotechnology and Protein Engineering
- Immunology and Complement System
- Pharmacology and Drug Development
Background:
- Aberrant complement regulation is implicated in diseases like C3 glomerulopathy (C3G) and dry age-related macular degeneration, with factor H (FH) as a potential therapeutic target.
- Microbial production of recombinant human FH (hFH) and murine FH (mFH) is desirable for high-dose therapies and pre-clinical research, respectively.
- Previous attempts using Pichia pastoris yielded modest or minuscule quantities of hFH and mFH, respectively.
Purpose of the Study:
- To enhance the microbial production of recombinant murine factor H (mFH) for pre-clinical studies.
- To investigate the therapeutic potential of recombinant mFH in a mouse model of C3 glomerulopathy.
- To optimize the production and characterization of recombinant mFH.
Main Methods:
- Engineered a Pichia pastoris strain overexpressing protein-disulfide isomerase (PDI) and a codon-modified gene for mFH under a methanol-inducible promoter.
- Optimized fermentation conditions, purified recombinant mFH, and enzymatically removed N-glycans.
- Administered de-glycosylated recombinant mFH to FH-knockout mice and assessed its pharmacokinetics, immunogenicity, and efficacy in reducing C3 deposition.
Main Results:
- The engineered Pichia pastoris strain achieved high yields of recombinant mFH (tens of mg/L), a significant improvement over previous methods.
- De-glycosylated recombinant mFH exhibited a shorter half-life and induced higher levels of anti-mFH antibodies compared to native mFH in mice.
- Despite rapid clearance, sequential administration of recombinant mFH significantly reduced C3 fragment deposition in the glomerular basement membrane, a hallmark of C3G.
Conclusions:
- Overexpression of PDI in Pichia pastoris is a highly effective strategy for boosting recombinant protein production, particularly for complex proteins like factor H.
- While de-glycosylation impacts mFH pharmacokinetics and immunogenicity, recombinant mFH demonstrates therapeutic potential in reducing complement deposition in C3G models.
- Further research is warranted to refine recombinant FH formulations for improved therapeutic efficacy and reduced immunogenicity in complement-mediated diseases.
