Dataset Information

Site-specific protein transamination using N-methylpyridinium-4-carboxaldehyde.

ABSTRACT: The controlled attachment of synthetic groups to proteins is important for a number of fields, including therapeutics, where antibody-drug conjugates are an emerging area of biologic medicines. We have previously reported a site-specific protein modification method using a transamination reaction that chemoselectively oxidizes the N-terminal amine of a polypeptide chain to a ketone or an aldehyde group. The newly introduced carbonyl can be used for conjugation to a synthetic group in one location through the formation of an oxime or a hydrazone linkage. To expand the scope of this reaction, we have used a combinatorial peptide library screening platform as a method to explore new transamination reagents while simultaneously identifying their optimal N-terminal sequences. N-Methylpyridinium-4-carboxaldehyde benzenesulfonate salt (Rapoport's salt, RS) was identified as a highly effective transamination reagent when paired with glutamate-terminal peptides and proteins. This finding establishes RS as a transamination reagent that is particularly well suited for antibody modification. Using a known therapeutic antibody, herceptin, it was demonstrated that RS can be used to modify the heavy chains of the wild-type antibody or to modify both the heavy and the light chains after N-terminal sequence mutation to add additional glutamate residues.

SUBMITTER: Witus LS

PROVIDER: S-EPMC4136391 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Site-specific protein transamination using N-methylpyridinium-4-carboxaldehyde.

Witus Leah S LS Netirojjanakul Chawita C Palla Kanwal S KS Muehl Ellen M EM Weng Chih-Hisang CH Iavarone Anthony T AT Francis Matthew B MB

Journal of the American Chemical Society 20131105 45

The controlled attachment of synthetic groups to proteins is important for a number of fields, including therapeutics, where antibody-drug conjugates are an emerging area of biologic medicines. We have previously reported a site-specific protein modification method using a transamination reaction that chemoselectively oxidizes the N-terminal amine of a polypeptide chain to a ketone or an aldehyde group. The newly introduced carbonyl can be used for conjugation to a synthetic group in one locatio ...[more]

PMID: 24191658

Similar Datasets

Project description:Site-specific protein labeling is an important technique in protein chemistry and is used for diverse applications ranging from creating protein conjugates to protein immobilization. Enzymatic reactions, including protein prenylation, have been widely exploited as methods to accomplish site-specific labeling. Enzymatic prenylation is catalyzed by prenyltransferases, including protein farnesyltransferase (PFTase) and geranylgeranyltransferase type I (GGTase-I), both of which recognize C-terminal CaaX motifs with different specificities and transfer prenyl groups from isoprenoid diphosphates to their respective target proteins. A number of isoprenoid analogues containing bioorthogonal functional groups have been used to label proteins of interest via PFTase-catalyzed reaction. In this study, we sought to expand the scope of prenyltransferase-mediated protein labeling by exploring the utility of rat GGTase-I (rGGTase-I). First, the isoprenoid specificity of rGGTase-I was evaluated by screening eight different analogues and it was found that those with bulky moieties and longer backbone length were recognized by rGGTase-I more efficiently. Taking advantage of the different substrate specificities of rat PFTase (rPFTase) and rGGTase-I, we then developed a simultaneous dual labeling method to selectively label two different proteins by using isoprenoid analogue and CaaX substrate pairs that were specific to only one of the prenyltransferases. Using two model proteins, green fluorescent protein with a C-terminal CVLL sequence (GFP-CVLL) and red fluorescent protein with a C-terminal CVIA sequence (RFP-CVIA), we demonstrated that when incubated together with both prenyltransferases and the selected isoprenoid analogues, GFP-CVLL was specifically modified with a ketone-functionalized analogue by rGGTase-I and RFP-CVIA was selectively labeled with an alkyne-containing analogue by rPFTase. By switching the ketone-containing analogue to an azide-containing analogue, it was possible to create protein tail-to-tail dimers in a one-pot procedure through the copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) reaction. Overall, with the flexibility of using different isoprenoid analogues, this system greatly extends the utility of protein labeling using prenyltransferases.

Dataset Information

Site-specific protein transamination using N-methylpyridinium-4-carboxaldehyde.

Publications

Site-specific protein transamination using N-methylpyridinium-4-carboxaldehyde.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets