Site-specific chemical protein conjugation using genetically encoded aldehyde tags

Abstract

We describe a method for modifying proteins site-specifically using a chemoenzymatic bioconjugation approach. Formylglycine generating enzyme (FGE) recognizes a pentapeptide consensus sequence, CxPxR, and it specifically oxidizes the cysteine in this sequence to an unusual aldehyde-bearing formylglyine. The FGE recognition sequence, or aldehyde tag, can be inserted into heterologous recombinant proteins produced in either prokaryotic or eukaryotic expression systems. The conversion of cysteine to formylglycine is accomplished by co-overexpression of FGE, either transiently or as a stable cell line, and the resulting aldehyde can be selectively reacted with α-nucleophiles to generate a site-selectively modified bioconjugate. This protocol outlines both the generation and the analysis of proteins aldehyde-tagged at their termini and the methods for chemical conjugation to the formylglycine. The process of generating aldehyde-tagged protein followed by chemical conjugation and purification takes 20 d.

Figure 1

Schemes illustrating our approach to chemical modification of proteins. (a) Conversion of cysteine to formylglycine within the FGE recognition peptide sequence. (b) Coexpressing an aldehyde-tagged protein with FGE results in a site-specifically modified protein with an aldehyde that can be subsequently modified with aldehyde-specific chemical cargo.

Figure 2

Anticipated results of LC-MS analysis of tryptic peptides from an aldehyde-tagged protein. Protein was reduced with DTT, alkylated with IAA and digested with trypsin. The sample was analyzed by LC-MS on a Waters QToF mass spectrometer. (a) Expected tag peptides. To determine conversion rates, we monitored the presence of ions corresponding to the peptides containing the formylglycine modification as the aldehyde (i) or the diol (ii) or the unconverted peptide with the Cys protected with iodoacetamide (iii). (b) Illustration of extracted ion currents (XIC) corresponding to the peptides indicated: (i), M ^{+ 2H} at m/z 693.9; (ii), M ^{+ 2H} at m/z 702.9; and (iii), M ^{+ 2H} at m/z 731.4. Note that the carbamidomethylated Cys-containing peptide elutes slightly later than its aldehyde-containing counterpart. (c) Shows mass spectra of the same peptides. The area under the curve (AUC) of the relevant XICs was used to determine the conversion rates as follows: i + ii/i + ii + iii.

Figure 3

Detection of conjugation: results expected when an aldehyde-tagged protein (LCTPSR) and the Cys-to-Ala control (LATPSR) from Step 27 are run on an SDS-PAGE gel. (a) The gel is stained with Sypro Orange to determine protein loading. (b) The fluorescence of the gel is measured to determine aldehyde-specific protein conjugation of an aldehyde-specific fluorophore to the LCTPSR-tagged protein; LATPSR-tagged protein is not converted to a formylglycine and will not be conjugated to the fluorophore.