Recombinant human serum amyloid P component from Pichia pastoris: production and characterization.
Human serum amyloid P component (SAP) was expressed in the methylotrophic yeast Pichia pastoris. SAP cDNA was placed under control of regulatory sequences derived from the alcohol oxidase gene (AOX1), and its protein product was secreted using the Saccharomyces cerevisiae alpha-mating factor signal sequence. Recombinant SAP (r-SAP) was produced in a bioreactor with computer controlled fed-batch mode and purified by use of a C-terminal histidine tag. The yield of purified r-SAP was 3-4mg from 1L supernatant and 5-6mg from 1L cell paste, indicating that the majority of the produced SAP was not secreted. Treatment of the cell paste with EDTA increased the yield further by about 30%. The N-terminal of r-SAP purified from the supernatant showed non-complete cleavage of the alpha-mating factor signal sequence. Purified r-SAP, analyzed under native conditions, was shown to be a decamer, like purified human SAP (h-SAP), with monomers of 27kDa. Each monomer had one N-glycosylation site, positioned at the same site as for h-SAP. r-SAP bound to antibodies produced against h-SAP. Furthermore, r-SAP bound to ds DNA and influenza A virus subunits in a Ca(2+)-dependent manner and inhibited influenza A virus hemagglutination. These results indicate that r-SAP produced in P. pastoris has the same biological activity as purified h-SAP.
Boysen
Susanne
S
Department of Immunology and Microbiology, Institute of Medical Biology, University of Southern Denmark, Winslowparken 21, DK-5000 Odense C, Denmark.
Fogh-Schultz
Berit
B
Andersen
Inger
I
Højrup
Peter
P
Iversen
Jens Jørgen Lønsmann
JJ
Wittenhagen
Per
P
Nielsen
Ellen Holm
EH
Svehag
Sven-Erik
SE
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D000683:Serum Amyloid P-Component / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification
D017931:DNA Primers; D011994:Recombinant Proteins; D000683:Serum Amyloid P-Component