Cysteine-containing fusion tag for site-specific conjugation of therapeutic and imaging agents to targeting proteins

Methods in Molecular Biology
Marina V BackerJoseph M Backer

Abstract

Targeted delivery of therapeutic and imaging agents requires conjugation of a corresponding payload to a targeting peptide or protein. The ideal procedure should yield a uniform preparation of functionally active conjugates and be translatable for development of clinical products. We describe here our experience with site-specific protein modification via a novel cysteine-containing fusion tag (Cys-tag), which is a 15-amino-acid (aa) N-terminal fragment of human ribonuclease I with the R4C substitution. Several Cys-tagged proteins and peptides with different numbers of native cysteines were expressed and refolded into functionally active conformation, indicating that the tag does not interfere with the formation of internal disulfide bonds. We also describe standardized procedures for site-specific conjugation of very different payloads, such as functionalized lipids and liposomes, radionuclide chelators and radionuclides, fluorescent dyes, drug-derivatized dendrimers, scaffold proteins, biotin, and polyethyleneglycol to Cys-tagged peptides and proteins, as well as present examples of functional activity of targeted conjugates in vitro and in vivo. We expect that Cys-tag would provide new opportunities for development of target...Continue Reading

Citations

May 8, 2010·Angiogenesis·T J A SnoeksE L Kaijzel
Aug 25, 2010·Investigative Radiology·Christopher R AndersonAlexander L Klibanov
Jun 28, 2012·Theranostics·Marina V Backer, Joseph M Backer
Jan 8, 2014·Theranostics·Irina Velikyan
Sep 6, 2011·Journal of Nuclear Medicine : Official Publication, Society of Nuclear Medicine·Francis G BlankenbergJoseph M Backer
Nov 17, 2009·Journal of Nuclear Medicine : Official Publication, Society of Nuclear Medicine·Zoia LevashovaFrancis G Blankenberg

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