Juárez-González V R, Riaño-Umbarila L, Quintero-Hernández V, Olamendi-Portugal T, Ortiz-León M, Ortíz E, Possani L D, Becerril B
Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, UNAM, Apartado Postal 510-3, Cuernavaca, Morelos 62250, Mexico.
J Mol Biol. 2005 Mar 11;346(5):1287-97. doi: 10.1016/j.jmb.2004.12.060. Epub 2005 Jan 16.
BCF2, a monoclonal antibody raised against scorpion toxin Cn2, is capable of neutralizing both, the toxin and the whole venom of the Mexican scorpion Centruroides noxius Hoffmann. The single chain antibody fragment (scFv) of BCF2 was constructed and expressed in Escherichia coli. Although its affinity for the Cn2 toxin was shown to be in the nanomolar range, it was non-neutralizing in vivo due to a low stability. In order to recover the neutralizing capacity, the scFv of BCF2 was evolved by error-prone PCR and the variants were panned by phage display. Seven improved mutants were isolated from three different libraries. One of these mutants, called G5 with one mutation at CDR1 and another at CDR2 of the light chain, showed an increased affinity to Cn2, as compared to the parental scFv. A second mutant, called B7 with a single change at framework 2 of heavy chain, also had a higher affinity. Mutants G5 and B7 were also improved in their stability but they were unable to neutralize the toxin. Finally, we constructed a variant containing the changes present in G5 and B7. The purpose of this construction was to combine the increments in affinity and stability borne by these mutants. The result was a triple mutant capable of neutralizing the Cn2 toxin. This variant showed the best affinity constant (KD=7.5x10(-11) M), as determined by surface plasmon resonance (BIAcore). The k(on) and k(off) were improved threefold and fivefold, respectively, leading to 15-fold affinity improvement. Functional stability determinations by ELISA in the presence of different concentrations of guanidinium hydrochloride (Gdn-HCl) revealed that the triple mutant is significantly more stable than the parental scFv. These results suggest that not only improving the affinity but also the stability of our scFv were important for recovering its neutralization capacity. These findings pave the way for the generation of recombinant neutralizing antisera against scorpion stings based on scFvs.
BCF2是一种针对蝎毒素Cn2产生的单克隆抗体，能够中和墨西哥蝎Centruroides noxius Hoffmann的毒素及全毒液。构建了BCF2的单链抗体片段（scFv）并在大肠杆菌中表达。尽管其对Cn2毒素的亲和力显示在纳摩尔范围内，但由于稳定性低，在体内无中和作用。为恢复中和能力，通过易错PCR对BCF2的scFv进行进化，并通过噬菌体展示淘选变体。从三个不同文库中分离出七个改进突变体。其中一个突变体称为G5，轻链的互补决定区1（CDR1）和CDR2各有一个突变，与亲本scFv相比，对Cn2的亲和力增加。另一个突变体称为B7，重链框架2有一个单一变化，也具有更高的亲和力。突变体G5和B7的稳定性也有所提高，但它们无法中和毒素。最后，构建了一个包含G5和B7中存在变化的变体。此构建的目的是将这些突变体带来的亲和力和稳定性增加结合起来。结果得到一个能够中和Cn2毒素的三重突变体。通过表面等离子体共振（BIAcore）测定，该变体显示出最佳亲和力常数（KD = 7.5×10^(-11) M）。正向速率常数（k(on)）和逆向速率常数（k(off)）分别提高了三倍和五倍，导致亲和力提高了15倍。在不同浓度盐酸胍（Gdn-HCl）存在下通过ELISA进行的功能稳定性测定表明，三重突变体比亲本scFv明显更稳定。这些结果表明，不仅提高scFv的亲和力，而且提高其稳定性对于恢复其中和能力都很重要。这些发现为基于scFv产生抗蝎蜇伤的重组中和抗血清铺平了道路。
