Purification and characterization of a recombinant rat prohaptoglobin expressed in baculovirus-infected Sf9 insect cells.
To generate hemoglobin-free full-length haptoglobin the cDNA encoding rat haptoglobin alphabeta subunits was cloned into shuttle vector pVT-Bac-His and used to produce a recombinant baculovirus Autographa californica Nuclear Polyhedrosis Virus (AcNPV) as an expression vector, named HpAcNPV. Recombinant virus was used to infect Spodoptera frugiperda (Sf9) insect cells. The 50 kDa protein expressed was mostly secreted into the culture medium at relatively high titer (15 microg/mL) and was found to be rat prohaptoglobin having a vector-derived N-terminal extension of 37 amino acids, containing both a hexahistidine tag and an enterokinase recognition sequence. The protein was successfully purified by a three step procedure including nickel-linked agarose and DEAE-Sepharose chromatography steps. Hemoglobin was not detected in the purified preparations. Purified recombinant rat prohaptoglobin protein was also found to be glycosylated, and to be capable of forming a complex with rat hemoglobin in vitro.
CIHR Group in Lung Development, Toronto, Ont., Canada M5G-1X8.
Alexander
A
Hillar
Gail
G
Otulakowski
Hugh
H
O Brodovich
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D018076:DNA, Complementary; D004797:Enzyme-Linked Immunosorbent Assay; D006242:Haptoglobins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006868:Hydrolysis; D016133:Polymerase Chain Reaction; D011498:Protein Precursors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera
D017931:DNA Primers; D018076:DNA, Complementary; D006242:Haptoglobins; D011498:Protein Precursors; D011994:Recombinant Proteins; C042034:prohaptoglobin