Chemoenzymatic Reversible Immobilization and Labeling of Proteins without Prior Purification

Abstract

Site-specific chemical modification of proteins is important for many applications in biology and biotechnology. Recently, our laboratory and others have exploited the high specificity of the enzyme protein farnesyltransferase (PFTase) to site-specifically modify proteins through the use of alternative substrates that incorporate bioorthogonal functionality including azides and alkynes. In this study, we evaluate two aldehyde-containing molecules as substrates for PFTase and as reactants in both oxime and hydrazone formation. Using green fluorescent protein (GFP) as a model system, we demonstrate that the purified protein can be enzymatically modified with either analogue to yield aldehyde-functionalized proteins. Oxime or hydrazone formation was then employed to immobilize, fluorescently label or PEGylate the resulting aldehyde-containing proteins. Immobilization via hydrazone formation was also shown to be reversible via transoximization with a fluorescent alkoxyamine. After characterizing this labeling strategy using pure protein, the specificity of the enzymatic process was used to selectively label GFP present in crude E. coli extract followed by capture of the aldehyde-modified protein using hydrazide-agarose. Subsequent incubation of the immobilized protein using a fluorescently labeled or PEGylated alkoxyamine resulted in the release of pure GFP containing the desired site-specific covalent modifications. This procedure was also employed to produce PEGylated glucose-dependent insulinotropic polypeptide (GIP), a protein with potential therapeutic activity for diabetes. Given the specificity of the PFTase-catalyzed reaction coupled with the ability to introduce a CAAX-box recognition sequence onto almost any protein, this method shows great potential as a general approach for the selective immobilization and labeling of recombinant proteins present in crude cellular extract without prior purification. Beyond generating site-specifically modified proteins, this approach for polypeptide modification could be particularly useful for large scale production of protein conjugates for therapeutic or industrial applications.

A Highly Efficient Catalyst for Oxime Ligation and Hydrazone–Oxime Exchange Suitable for Bioconjugation

“…In previous work, we reported on a method for site-specifically functionalizing a protein in a crude cell mixture, and labeling and purifying the modified protein using aniline without any additional purification. 18 Although that process was successful, it required long reaction times to achieve significant levels of conversion. Accordingly, we next re-evaluated our aforementioned method for protein labeling with the new, more effective, mPDA catalyst discovered here.…”

“…Among all imine-based reactions, oximes are the most stable imines 10 and as a result have found widespread use in applications such as protein labeling, 11–15 analyzing protein-protein interactions and in vivo cell imaging. 11, 12 Although aniline is known to catalyze the oxime formation reaction, it suffers from relatively slow reaction kinetics, especially when a ketone is being used 11,18 or when transoximization is conducted. 13 In the case of ketones under physiological conditions, even in presence of aniline, it takes several hours for the reaction to be complete although faster rates can be achieved if higher reactant concentrations are employed.…”

A Pictet-Spengler ligation for protein chemical modification

“…Several chemical, enzymatic, and genetic methods have been developed to introduce aldehydes and ketones into proteins site specifically. These include periodate oxidation of N-terminal serine or threonine residues (9), pyridoxal phosphate-mediated N-terminal transamination to yield an α-ketoamide or glyoxamide (10-13), addition of ketone-containing small molecules to protein C-terminal thioesters generated by expressed protein ligation (14), genetically encoded incorporation of unnatural amino acids containing ketones via amber stop codon suppression (15)(16)(17)(18), genetic encoding of peptide tags that direct enzymatic ligation of aldehyde-or ketonebearing small molecules (19,20), and genetic encoding of a site for modification by the formylglycine-generating enzyme (FGE), the "aldehyde tag" method developed in our laboratory (21)(22)(23)(24)(25).…”

Efficient farnesylation of an extended C-terminal C(x)3X sequence motif expands the scope of the prenylated proteome

“…Following eGFP-CAVGP incubation with C15AlkOPP in the presence of FTase, the modified protein was derivatized with TAMRA-N3 using Cu(II)-catalyzed alkyne-azide cycloaddition (CuAAC). (71,72) In-gel imaging of TAMRA fluorescence revealed a single band at the expected size of farnesylated eGFP-GCAVGP (~28 kDa, Figure 4c). The negative control reaction lacking the alkyne FPP analogue does not exhibit a TAMRA-fluorescent band, with Coomassie Blue staining confirming the presence of eGFP-CAVGP in both reactions.…”