Synthesis of peptide-protein conjugates using N-succinimidyl carbamate chemistry.
Peptide-protein conjugates are useful tools in different fields of research as, for instance, the development of vaccines and drugs or for studying biological mechanisms, to cite only few applications. N-Succinimidyl carbamate (NSC) chemistry has been scarcely used in this area. We show that unprotected peptides, featuring one lysine residue within their sequences, can be converted in good yield into NSC derivatives by reaction with disuccinimidylcarbonate (DSC). No hydrolysis of the NSC group was observed during RP-HPLC purification, lyophilization, or storage. NSC peptides reacted efficiently within minutes with lysozyme used as model protein. To illustrate usefulness of the method consisting of the synthesis of a peptide-protein conjugate of biological interest, a NSC peptide derived from a peptide substrate for tyrosylprotein sulfotransferase (TS) was synthesized and ligated to receptor-binding nontoxic B-subunit of Shiga toxin (STxB). Immunofluorescence studies showed the intracellular delivery of the TS-STxB conjugate and its ability to circulate to the Golgi as the native STxB protein. Moreover, we demonstrate that the TS label could be sulfated by tyrosylprotein sulfotransferases present in the Golgi. Thus, NSC chemistry permitted rapid synthesis of a peptide-protein conjugate worthwhile for studying the transport of proteins from the plasma membrane to the Golgi. The second part of this article describes a more general method for synthesizing peptide-protein conjugates without any limitation of the peptide sequence. The conjugates were assembled by combining NSC chemistry and alpha-oxo semicarbazone ligation. To this end, a glyoxylyl NSC peptide was synthesized and reacted with lysozyme. The glyoxylyl groups on the protein were then reacted with a semicarbazide peptide to produce the target peptide-protein conjugate. Both reactions, namely, urea bond formation and alpha-oxo semicarbazone ligation, were carried at pH 8.0 using a one-pot procedure.
Mhidia
Reda
R
UMR CNRS 8161 Universite de Lille Nord de France, Institut Pasteur de Lille, IFR 142, 1 rue du Pr Calmette 59021 Lille Cedex, France.
Vallin
Aurélie
A
Ollivier
Nathalie
N
Blanpain
Annick
A
Shi
Getao
G
Christiano
Romain
R
Johannes
Ludger
L
Melnyk
Oleg
O
D000595:Amino Acid Sequence; D002219:Carbamates / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009113:Muramidase / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis* / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D055550:Protein Stability; D021381:Protein Transport; D011506:Proteins / Q000138:chemical synthesis* / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D013386:Succinates / Q000737:chemistry*
D002219:Carbamates; D010455:Peptides; D011506:Proteins; D013386:Succinates; D009113:Muramidase