Quadros E V, Sai P, Rothenberg S P
Division of Hematology/Oncology, SUNY-Health Science Center, Brooklyn.
Blood. 1993 Mar 1;81(5):1239-45.
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.
转钴胺素II（TCII）是哺乳动物血浆中一种钴胺素（Cbl，维生素B12）结合蛋白，可促进细胞对该维生素的摄取。为了获得足够量的人TCII用于分析研究，将编码TCII的互补DNA（cDNA）插入质粒PVL 1393中，并通过与野生型病毒共转染，在草地贪夜蛾（SF9）昆虫细胞中通过同源重组获得表达TCII的杆状病毒。在优化条件下，感染重组病毒的SF9细胞每毫升培养基分泌2至4微克TCII。TCII不会在SF9细胞中积累，并且似乎如先前在培养的人内皮细胞中观察到的那样持续分泌。通过SDS-PAGE分析，纯化的重组TCII与纯化的人TCII具有相同的分子量。重组TCII与抗天然人TCII的抗血清发生交叉反应，结合Cbl，并通过与K562细胞膜上的TCII-Cbl受体结合，促进真核细胞对Cbl的摄取。对纯化的重组TCII进行氨基酸序列分析，鉴定出两种多肽，一种与从cDNA推导的氨基酸序列相同，另一种缺少前两个N端残基。这些序列与从人混合血浆的Cohn组分III中纯化的两种TCII多肽相同。重组TCII的两种形式与人类血清中TCII的两种主要同蛋白形式具有相同的等电点。由于杆状病毒构建体包含一个只能编码一个氨基酸序列的单一cDNA，重组TCII中的两种同蛋白必定是通过等位基因特异性表达以外的机制产生的。产生非糖基化肽（如TCII）同蛋白的一种合理机制可能是前导肽在替代位点的剪接。
