PROTEIN ENGINEERING OF ANTIBODY-BINDING SITES - RECOVERY OF SPECIFIC ACTIVITY IN AN ANTI-DIGOXIN SINGLE-CHAIN FV ANALOG PRODUCED IN ESCHERICHIA-COLI
PROTEIN ENGINEERING OF ANTIBODY-BINDING SITES - RECOVERY OF SPECIFIC ACTIVITY IN AN ANTI-DIGOXIN SINGLE-CHAIN FV ANALOG PRODUCED IN ESCHERICHIA-COLI
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DOI：
10.1073/pnas.85.16.5879
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发表时间：
1988-08-01
影响因子：
11.1
通讯作者：
OPPERMANN, H
中科院分区：
文献类型：
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作者：
HUSTON, JS;LEVINSON, D;OPPERMANN, H
通过蛋白质工程在大肠杆菌中产生生物合成抗体结合位点，其将抗地高辛单克隆抗体26 - 10的可变结构域并入单个多肽链（Mr = 26，354）中。该可变区片段（Fv）类似物包含通过15-氨基辅助接头连接的26 - 10个重链和轻链可变区（VH和VL）以形成单链Fv（sFV）。sFv被设计为248个氨基酸的脯氨酰-VH-（接头）-VL序列。用E. coli密码子偏好性，并从合成的寡核苷酸开始组装sFv基因。在E. coli中，使用源自trp LE序列的前导序列。sFv蛋白通过在融合蛋白[（leader）-Asp-Pro-VH-（linker）-VL]中前导序列和sFv的连接处改造的独特Asp-Pro肽键的酸切割获得。分离和复性后，折叠的sFv显示出与天然26 - 10 Fab片段相似的地高辛和相关强心苷的特异性。亲和纯化的sFv和地高辛之间的结合表现出缔合常数[Ka =（3.2 ± 0.01）]。0.9). times. 107 M-1]，其比26 - 10 Fab片段[Ka =（1.9. ±. 0.2). times. 108 M-1]在相同的缓冲液条件下，由0.01 M乙酸钠，pH 5.5/0.25 M尿素组成。
A biosynthetic antibody binding site, which incorporated the variable domains of anti-digoxin monoclonal antibody 26-10 in a single polypeptide chain (Mr = 26,354), was produced in Escherichia coli by protein engineering. This variable region fragment (Fv) analogue comprised the 26-10 heavy- and light-chain variable regions (VH and VL) connected by a 15-amino aid linker to form a single-chain Fv (sFV). The sFv was designed as a prolyl-VH-(linker)-VL sequence of 248 amino acids. A 744-base-pair DNA sequence corresponding to this sFv protein was derived by using an E. coli codon preference, and the sFv gene was assembled starting from synthetic oligonucleotides. The sFv polypeptide was expressed as a fusion protein in E. coli, using a leader derived from the trp LE sequence. The sFv protein was obtained by acid cleavage of the unique Asp-Pro peptide bond engineered at the junction of leader and sFv in the fusion protein [(leader)-Asp-Pro-VH-(linker)-VL]. After isolation and renaturation, folded sFv displayed specificity for digoxin and related cardiac glycosides similar to that of natural 26-10 Fab fragments. Binding between affinity-purified sFv and digoxin exhibited an association constant [Ka = (3.2 .+-. 0.9) .times. 107 M-1] that was about a factor of 6 smaller than that found for 26-10 Fab fragments [Ka = (1.9 .+-. 0.2) .times. 108 M-1] under the same buffer conditions, consisting of 0.01 M sodium acetate, pH 5.5/0.25 M urea.