McCartney J E, Lederman L, Drier E A, Cabral-Denison N A, Wu G M, Batorsky R S, Huston J S, Oppermann H
Creative BioMolecules, Inc., Hopkinton, Massachusetts 01748.
J Protein Chem. 1991 Dec;10(6):669-83. doi: 10.1007/BF01025718.
The functional antigen binding region of antidinitrophenol mouse IgA myeloma MOPC 315 has been produced as a single-chain Fv (sFv) protein in E. coli. Recombinant 315 proteins included sFv alone, a bifunctional fusion protein with amino-terminal fragment B (FB) of staphylococcal protein A, and a two-chain 315 Fv fragment. Successful refolding of the 315 sFv required formation of disulfide bonds while the polypeptide was in a denatured state, as previously observed for the parent Fv fragment. Affinity-purified recombinant 315 proteins showed full recovery of specific activity, with values for Ka,app of 1.5 to 2.2 x 10(6) M-1, equivalent to the parent 315 Fv fragment. As observed for natural 315 Fv, the sFv region of active FB-sFv315 fusion protein was resistant to pepsin treatment, whereas inactive protein was readily degraded. These experiments will allow the application of protein engineering to the 315 single-chain Fv; such studies can advance structure-function studies of antibody combining sites and lead to an improved understanding of single-chain Fv proteins.
抗二硝基苯酚小鼠IgA骨髓瘤MOPC 315的功能性抗原结合区已在大肠杆菌中作为单链Fv（sFv）蛋白产生。重组315蛋白包括单独的sFv、与葡萄球菌蛋白A的氨基末端片段B（FB）的双功能融合蛋白以及双链315 Fv片段。315 sFv的成功重折叠需要在多肽处于变性状态时形成二硫键，这与之前观察到的亲本Fv片段情况相同。亲和纯化的重组315蛋白显示出比活性的完全恢复，表观解离常数（Ka,app）值为1.5至2.2×10(6) M-1，与亲本315 Fv片段相当。正如天然315 Fv所观察到的那样，活性FB-sFv315融合蛋白的sFv区域对胃蛋白酶处理具有抗性，而无活性的蛋白则很容易被降解。这些实验将使蛋白质工程能够应用于315单链Fv；此类研究可以推进抗体结合位点的结构-功能研究，并有助于更好地理解单链Fv蛋白。
