LPS ligand and culture additives improve production of monomeric MD-1 and 2 in Pichia pastoris by decreasing aggregation and intermolecular disulfide bonding.
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.
Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom.
Kristen E
KE
Mengwasser
Clare E
CE
Bryant
Nick J
NJ
Gay
Monique
M
Gangloff
D000818:Animals; D000954:Antigens, Surface / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003470:Culture Media; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006736:Horses; D006801:Humans; D008024:Ligands; D008070:Lipopolysaccharides / Q000494:pharmacology*; D051218:Lymphocyte Antigen 96 / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D010641:Phenotype; D010843:Pichia / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D000954:Antigens, Surface; D003470:Culture Media; D004220:Disulfides; C115213:LY86 protein, human; C495377:LY96 protein, human; D008024:Ligands; D008070:Lipopolysaccharides; D051218:Lymphocyte Antigen 96; D011993:Recombinant Fusion Proteins