Functional human transcobalamin II isoproteins are secreted by insect cells using the baculovirus expression system
Functional human transcobalamin II isoproteins are secreted by insect cells using the baculovirus expression system
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昆虫细胞使用杆状病毒表达系统分泌功能性人转钴胺素 II 同种蛋白
DOI：
10.1182/blood.v81.5.1239.1239
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发表时间：
1993
期刊：
影响因子：
20.3
通讯作者：
S. Rothenberg
中科院分区：
文献类型：
--
作者：
E. Quadros;P. Sai;S. Rothenberg
转钴胺素II（TCII）是哺乳动物血浆中的钴胺素（Cbl，维生素B12）结合蛋白，可促进维生素的细胞摄取。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（Spodoptera frugiperda，SF 9）昆虫细胞中同源重组获得表达TCII的杆状病毒。在优化的条件下，用重组病毒感染的SF 9细胞每毫升培养基分泌2至4微克TCII。TCII没有积累在SF 9细胞中，似乎是组成性分泌，如先前在培养的人内皮细胞中观察到的。纯化的重组TCII通过SDS-PAGE具有与纯化的人TCII相同的分子量。重组TCII与天然人TCII的抗血清交叉反应，结合Cbl并通过结合K562细胞质膜上的TCII-Cbl受体促进真核细胞中Cbl的摄取。纯化的重组TCII的氨基酸序列分析鉴定了两种多肽，一种与从cDNA推导的氨基酸序列相同，第二种缺乏第一和第二N-末端残基。这些序列与从合并的人血浆的Cohn组分III纯化的两种TCII多肽相同。两种形式的重组TCII具有与人血清中TCII的两种主要同种蛋白形式相同的等电点。由于杆状病毒构建体含有只能编码一个氨基酸序列的单一cDNA，因此重组TCII中的两种同工蛋白必须通过等位基因特异性表达以外的机制产生。产生非糖基化肽（如TCII）的同工蛋白的合理机制可能是通过在替代位点剪接前导肽。
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.
人转钴胺素 II 的纯化和分子表征。
DOI：
--
发表时间：
1986
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Quadros,EV;Rothenberg,SP;Pan,YC;Stein,S
通讯作者：
Stein,S
人转钴胺素II的cDNA序列和推导的氨基酸序列与大鼠内因子和人转钴胺素I具有同源性。
DOI：
--
发表时间：
1991
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Platica,O;Janeczko,R;Quadros,EV;Regec,A;Romain,R;Rothenberg,SP
通讯作者：
Rothenberg,SP