LPS ligand and culture additives improve production of monomeric MD-1 and 2 in Pichia pastoris by decreasing aggregation and intermolecular disulfide bonding
| Autor: | Kristen E. Mengwasser, Clare E. Bryant, Nick J. Gay, Monique Gangloff |
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| Přispěvatelé: | Bryant, Clare [0000-0002-2924-0038], Gay, Nicholas [0000-0002-2782-7169], Gangloff, Monique [0000-0001-6131-0115], Apollo - University of Cambridge Repository |
| Jazyk: | angličtina |
| Předmět: |
Lipopolysaccharides
Recombinant Fusion Proteins Lymphocyte Antigen 96 Ligands Article Pichia Pichia pastoris chemistry.chemical_compound Aggregation Immune system Disulfide bonding Animals Humans Disulfides Horses Cloning Molecular Disulfide bonds Receptor Additive screening Expression vector biology Intermolecular force biology.organism_classification Culture Media Phenotype Monomer MD-1 chemistry Biochemistry MD-2 Antigens Surface Chromatography Gel TLR4 Electrophoresis Polyacrylamide Gel Pichia pastoris expression Protein Multimerization LPS binding Biotechnology |
| Zdroj: | Protein Expression and Purification |
| ISSN: | 1046-5928 |
| DOI: | 10.1016/j.pep.2010.11.018 |
| Popis: | Myeloid differentiation proteins MD-1 and MD-2 have both been shown to form a heterogeneous collection of oligomers when expressed in absence of their respective receptor, RP105 and TLR4. The biological relevance of these oligomers is not clear. Only monomeric proteins have been found to be active and able to trigger an immune response to endotoxin by modulating the TLR4 pathway. In this study, we produced variants of MD-1 and MD-2 in Pichia pastoris. To minimize the time and expense of initial expression tests, small-scale cultures have been set up to allow the rapid identification of the highest expressing clone and the optimal expression conditions. The expression vectors used, the site of linearization and the locus of integration affected the yield of transformation. Next we screened culture additives and found that they significantly increased the fraction of monomeric proteins secreted in the culture medium (up to 15% of the total MD protein produced). We confirmed their presence by size-exclusion chromatography. Optimal anti-aggregation agents were protein-dependent except for LPS that presented stabilizing effects for all MD proteins. Contrary to previous reports, this study suggests that MD-1 can bind to LPS. |
| Databáze: | OpenAIRE |
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