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Methods Protoc. 2018, 1(1), 2; https://doi.org/10.3390/mps1010002

Predictable Peptide Conjugation Ratios by Activation of Proteins with Succinimidyl Iodoacetate (SIA)

1
Federal Institute for Materials Research and Testing (BAM), Division 1.5 Protein Analysis, Richard-Willstätter-Strasse 11, 12489 Berlin, Germany
2
Humboldt-Universität zu Berlin, School of Analytical Sciences Adlershof, Unter den Linden 6, 10099 Berlin, Germany
3
Humboldt-Universität zu Berlin, Department of Chemistry, Brook-Taylor-Str. 2, 12489 Berlin, Germany
*
Author to whom correspondence should be addressed.
Academic Editor: Fernando Albericio
Received: 2 August 2017 / Revised: 12 September 2017 / Accepted: 14 September 2017 / Published: 25 September 2017
(This article belongs to the Special Issue Feature Papers)
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Abstract

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 N-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 in vivo. 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. View Full-Text
Keywords: bioconjugate; carrier protein; immunogen; hapten; linker; antibody drug conjugate (ADC); drug-to-antibody ratio (DAR); conjugation ratio; conjugation density; carrier load; click reaction bioconjugate; carrier protein; immunogen; hapten; linker; antibody drug conjugate (ADC); drug-to-antibody ratio (DAR); conjugation ratio; conjugation density; carrier load; click reaction
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).
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Abbas, I.M.; Schwaar, T.; Bienwald, F.; Weller, M.G. Predictable Peptide Conjugation Ratios by Activation of Proteins with Succinimidyl Iodoacetate (SIA). Methods Protoc. 2018, 1, 2.

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