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PMID:8443384

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Citation

Quadros, EV, Sai, P and Rothenberg, SP (1993) Functional human transcobalamin II isoproteins are secreted by insect cells using the baculovirus expression system. Blood 81:1239-45

Abstract

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.

Links

PubMed

Keywords

Amino Acid Sequence; Animals; Baculoviridae/genetics; Cell Line; Isoelectric Focusing; Molecular Sequence Data; Moths; Recombinant Proteins/biosynthesis; Transcobalamins/biosynthesis; Transfection

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

HUMAN:TCO2

located_in

GO:0005615: extracellular space

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

HUMAN:TCO2

involved_in

GO:0015889: cobalamin transport

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

HUMAN:TCO2

enables

GO:0031419: cobalamin binding

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

HUMAN:TCO2

part_of

GO:0005615: extracellular space

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

Notes

See also

References

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