A bifunctional fusion protein containing Fc-binding fragment B of staphylococcal protein A amino terminal to antidigoxin single-chain Fv.
A bifunctional fusion protein containing Fc-binding fragment B of staphylococcal protein A amino terminal to antidigoxin single-chain Fv.
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DOI：
10.1021/bi00487a005
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发表时间：
1990-09
期刊：
影响因子：
2.9
通讯作者：
M. Tai;Meredith Mudgett-Hunter;Douglas Levinson;Gay‐May Wu;Edgar Haber;Hermann Oppermann;James S. Huston
中科院分区：
文献类型：
--
作者：
M. Tai;Meredith Mudgett-Hunter;Douglas Levinson;Gay‐May Wu;Edgar Haber;Hermann Oppermann;James S. Huston
一种双功能分子经过基因工程改造，包含结合免疫球蛋白 Fc（葡萄球菌蛋白 A 的片段 B）的氨基末端效应结构域和结合地高辛 [单链 Fv (sFv)] 的羧基末端结构域。尽管 FB 靠近 sFv 结合位点，但效应子和 sFv 结合特性实际上与母体分子相同。这一发现是前所未有的，因为在天然免疫球蛋白超家族的所有分子中，抗原结合结构域位于效应结构域的氨基末端。 FB-sFv 序列由单个合成基因编码，并在大肠杆菌中表达为分子量为 33,106 的蛋白质。经过纯化、复性和亲和分离后，活性融合蛋白的产量为110mg/L发酵细胞（18.5g细胞糊）。通过平板测定和 Ouchterlony 分析测量，FB-sFv 将 IgG 与地高辛-牛血清白蛋白交联的能力证实了双功能。对表达的融合蛋白的分析表明，sFv 有望开发多功能、可靶向的单链蛋白。
A bifunctional molecule was genetically engineered which contained an amino-terminal effector domain that bound immunoglobulin Fc (fragment B of staphylococcal protein A) and a carboxyl-terminal domain that bound digoxin [a single-chain Fv (sFv)]. Effector and sFv binding properties were virtually identical with those of the parent molecules, despite the proximity of the FB to the sFv combining site. This finding is unprecedented since in all molecules of the natural immunoglobulin superfamily, the antigen binding domain is amino terminal to the effector domain. The FB-sFv sequence was encoded in a single synthetic gene and expressed as a 33,106 molecular weight protein in Escherichia coli. After purification, renaturation, and affinity isolation, yield of active fusion protein were 110 mg/L of fermented cells (18.5-g cell paste). Bifunctionality was confirmed by the ability of FB-sFv to cross-link IgG to digoxin-bovine serum albumin, as measured by plate assays and by Ouchterlony analysis. Analysis of the expressed fusion protein suggests that the sFv holds promise for the development of multifunctional, targetable single-chain proteins.