Abbas Ioana M, Schwaar Timm, Bienwald Frank, Weller Michael G
Federal Institute for Materials Research and Testing (BAM), Division 1.5 Protein Analysis, Richard-Willstätter-Strasse 11, 12489 Berlin, Germany.
Humboldt-Universität zu Berlin, School of Analytical Sciences Adlershof, Unter den Linden 6, 10099 Berlin, Germany.
Methods Protoc. 2017 Sep 25;1(1):2. doi: 10.3390/mps1010002.
The small heterobifunctional linker succinimidyl iodoacetate (SIA) was examined for the preparation of peptide-protein bioconjugates with predicable conjugation ratios. For many conjugation protocols, the protein is either treated with a reductant to cleave disulfide bonds or is reacted with thiolation chemicals, such as Traut's reagent. Both approaches are difficult to control, need individual optimization and often lead to unsatisfactory results. In another popular approach, a heterobifunctional linker with a -hydroxysuccinimide (NHS) and a maleimide functionality is applied to the protein. After the activation of some lysine ε-amino groups with the NHS ester functionality, a cysteine-containing peptide is attached to the activated carrier protein via maleimide. Particularly, the maleimide reaction leads to some unwanted byproducts or even cleavage of the linker. Many protocols end up with conjugates with unpredictable and irreproducible conjugation ratios. In addition, the maleimide-thiol addition product should be assumed immunogenic . To avoid these and other disadvantages of the maleimide approach, we examined the known linker succinimidyl iodoacetate (SIA) in more detail and developed two protocols, which lead to peptide-protein conjugates with predefined average conjugation ratios. This holds potential to eliminate tedious and expensive optimization steps for the synthesis of a bioconjugate of optimal composition.
研究了小分子异双功能连接子碘乙酸琥珀酰亚胺酯（SIA）用于制备具有可预测偶联比的肽 - 蛋白质生物共轭物。对于许多偶联方案，蛋白质要么用还原剂处理以裂解二硫键，要么与硫醇化化学试剂（如Traut试剂）反应。这两种方法都难以控制，需要单独优化，并且常常导致不理想的结果。在另一种常用方法中，将具有N - 羟基琥珀酰亚胺（NHS）和马来酰亚胺官能团的异双功能连接子应用于蛋白质。在用NHS酯官能团活化一些赖氨酸ε - 氨基后，含半胱氨酸的肽通过马来酰亚胺连接到活化的载体蛋白上。特别是，马来酰亚胺反应会产生一些不需要的副产物，甚至导致连接子断裂。许多方案最终得到的共轭物具有不可预测和不可重复的偶联比。此外，应假定马来酰亚胺 - 硫醇加成产物具有免疫原性。为避免马来酰亚胺方法的这些及其他缺点，我们更详细地研究了已知的连接子碘乙酸琥珀酰亚胺酯（SIA），并开发了两种方案，可得到具有预定义平均偶联比的肽 - 蛋白质共轭物。这有可能消除合成最佳组成生物共轭物时繁琐且昂贵的优化步骤。
