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Functional human transcobalamin II isoproteins are secreted by insect cells
using the baculovirus expression system
EV Quadros, P Sai and SP Rothenberg
Division of Hematology/Oncology, SUNY-Health Science Center, Brooklyn.
Transcobalamin II (TCII) is a cobalamin (Cbl, vitamin B12)-binding protein
in mammalian plasma that facilitates the cellular uptake of the vitamin. To
obtain human TCII in sufficient quantity for analytical studies, the
complementary DNA (cDNA) encoding TCII was inserted into the plasmid PVL
1393, and the baculovirus expressing TCII was obtained by homologous
recombination in Spodoptera frugiperda (SF9) insect cells by cotransfection
with the wildtype virus. Under optimized conditions, SF9 cells infected
with the recombinant virus secreted 2 to 4 micrograms of TCII per
milliliter of culture medium. TCII did not accumulate in the SF9 cells and
seemed to be constitutively secreted as observed previously in cultured
human endothelial cells. The purified recombinant TCII has the same
molecular weight by SDS-PAGE as purified human TCII. The recombinant TCII
cross-reacts with an antiserum to native human TCII, binds Cbl and
facilitates the uptake of Cbl in eukaryotic cells by binding to the
receptor for TCII-Cbl on the plasma membrane of K562 cells. Amino acid
sequence analysis of the purified recombinant TCII identified two
polypeptides, one identical to the amino acid sequence deduced from the
cDNA and a second lacking the first and second N-terminal residues. These
sequences are identical to two TCII polypeptides purified from Cohn
fraction III of pooled human plasma. The two forms of recombinant TCII have
the same isoelectric points as the two predominant isoprotein forms of TCII
in human serum. Since the baculovirus construct contains a single cDNA that
can encode only one amino acid sequence, the two isoproteins in recombinant
TCII must be generated by a mechanism other than allele specific
expression. A plausible mechanism for generating isoproteins of
nonglycosylated peptides, such as TCII, may be by splicing of the leader
peptide at alternative sites.
Volume 81,
Issue 5,
pp. 1239-1245,
03/01/1993
Copyright © 1993 by The American Society of Hematology
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