The site‐selective functionalization of proteins has broad application in chemical biology, but can be limited when mixtures result from incomplete conversion or the formation of protein containing side products. It is shown here that when proteins are covalently tagged with pyridyl‐tetrazines, the nickel‐iminodiacetate (Ni‐IDA) resins commonly used for His‐tags can be directly used for protein affinity purification. These Affinity Bioorthogonal Chemistry (ABC) tags serve a dual role by enabling affinity‐based protein purification while maintaining rapid kinetics in bioorthogonal reactions. ABC‐tagging works with a range of site‐selective bioconjugation methods with proteins tagged at the C‐terminus, N‐terminus or at internal positions. ABC‐tagged proteins can also be purified from complex mixtures including cell lysate. The combination of site‐selective conjugation and clean‐up with ABC‐tagged proteins also allows for facile on‐resin reactions to provide protein‐protein conjugates.
The site‐selective functionalization of proteins has broad application in chemical biology, but can be limited when mixtures result from incomplete conversion or the formation of protein containing side products. It is shown here that when proteins are covalently tagged with pyridyl‐tetrazines, the nickel‐iminodiacetate (Ni‐IDA) resins commonly used for His‐tags can be directly used for protein affinity purification. These Affinity Bioorthogonal Chemistry (ABC) tags serve a dual role by enabling affinity‐based protein purification while maintaining rapid kinetics in bioorthogonal reactions. ABC‐tagging works with a range of site‐selective bioconjugation methods with proteins tagged at the C‐terminus, N‐terminus or at internal positions. ABC‐tagged proteins can also be purified from complex mixtures including cell lysate. The combination of site‐selective conjugation and clean‐up with ABC‐tagged proteins also allows for facile on‐resin reactions to provide protein‐protein conjugates.
more » « less- NSF-PAR ID:
- 10379477
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Angewandte Chemie
- Volume:
- 134
- Issue:
- 45
- ISSN:
- 0044-8249
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
more » « less
Abstract -
more » « less
Abstract Class II major histocompatibility complex peptide (MHC‐IIp) multimers are precisely engineered reagents used to detect T cells specific for antigens from pathogens, tumors, and self‐proteins. While the related Class I MHC/peptide (MHC‐Ip) multimers are usually produced from subunits expressed in
E. coli , most Class II MHC alleles cannot be produced in bacteria, and this has contributed to the perception that MHC‐IIp reagents are harder to produce. Herein, we present a robust constitutive expression system for soluble biotinylated MHC‐IIp proteins that uses stable lentiviral vector−transduced derivatives of HEK‐293T cells. The expression design includes allele‐specific peptide ligands tethered to the amino‐terminus of the MHC‐II β chain via a protease‐cleavable linker. Following cleavage of the linker, HLA‐DM is used to catalyze efficient peptide exchange, enabling high‐throughput production of many distinct MHC‐IIp complexes from a single production cell line. Peptide exchange is monitored using either of two label‐free methods, native isoelectric focusing gel electrophoresis or matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometry of eluted peptides. Together, these methods produce MHC‐IIp complexes that are highly homogeneous and that form the basis for excellent MHC‐IIp multimer reagents. © 2021 Wiley Periodicals LLC.This article was corrected on 19 July 2022. See the end of the full text for details.
Basic Protocol 1 : Lentivirus production and expression line creationSupport Protocol 1 : Six‐well assay for estimation of production cell line yieldSupport Protocol 2 : Universal ELISA for quantifying proteins with fused leucine zippers and His‐tagsBasic Protocol 2 : Cultures for production of Class II MHC proteinsBasic Protocol 3 : Purification of Class II MHC proteins by anti‐leucine zipper affinity chromatographyAlternate Protocol 1 : IMAC purification of His‐tagged Class II MHCSupport Protocol 3 : Protein concentration measurements and adjustmentsSupport Protocol 4 : Polishing purification by anion‐exchange chromatographySupport Protocol 5 : Estimating biotinylation percentage by streptavidin precipitationBasic Protocol 4 : Peptide exchangeBasic Protocol 5 : Analysis of peptide exchange by matrix‐assisted laser desorption/ionization (MALDI) mass spectrometryAlternate Protocol 2 : Native isoelectric focusing to validate MHC‐II peptide loadingBasic Protocol 6 : MultimerizationBasic Protocol 7 : Staining cells with Class II MHC tetramers -
more » « less
Abstract Protein labeling strategies have been explored for decades to study protein structure, function, and regulation. Fluorescent labeling of a protein enables the study of protein‐protein interactions through biophysical methods such as microscale thermophoresis (MST). MST measures the directed motion of a fluorescently labeled protein in response to microscopic temperature gradients, and the protein's thermal mobility can be used to determine binding affinity. However, the stoichiometry and site specificity of fluorescent labeling are hard to control, and heterogeneous labeling can generate inaccuracies in binding measurements. Here, we describe an easy‐to‐apply protocol for high‐stoichiometric, site‐specific labeling of a protein at its N‐terminus with
N ‐hydroxysuccinimide (NHS) esters as a means to measure protein‐protein interaction affinity by MST. This protocol includes guidelines for NHS ester labeling, fluorescent‐labeled protein purification, and MST measurement using a labeled protein. As an example of the entire workflow, we additionally provide a protocol for labeling a ubiquitin E3 enzyme and testing ubiquitin E2‐E3 enzyme binding affinity. These methods are highly adaptable and can be extended for protein interaction studies in various biological and biochemical circumstances. © 2021 Wiley Periodicals LLC.This article was corrected on 18 July 2022. See the end of the full text for details.
Basic Protocol 1 : Labeling a protein of interest at its N‐terminus with NHS esters through stepwise reactionAlternate Protocol : Labeling a protein of interest at its N‐terminus with NHS esters through a one‐pot reactionBasic Protocol 2 : Purifying the N‐terminal fluorescent‐labeled protein and determining its concentration and labeling efficiencyBasic Protocol 3 : Using MST to determine the binding affinity of an N‐terminal fluorescent‐labeled protein to a binding partner.Basic Protocol 4 : NHS ester labeling of ubiquitin E3 ligase WWP2 and measurement of the binding affinity between WWP2 and an E2 conjugating enzyme by the MST binding assay -
more » « less
Rationale Purification of recombinant proteins is a necessary step for functional or structural studies and other applications. Immobilized metal affinity chromatography is a common recombinant protein purification method. Mass spectrometry (MS) allows for confirmation of identity of expressed proteins and unambiguous detection of enzymatic substrates and reaction products. We demonstrate the detection of enzymes purified on immobilized metal affinity surfaces by direct or ambient ionization MS, and follow their enzymatic reactions by direct electrospray ionization (ESI) or desorption electrospray ionization (DESI).
Methods A protein standard, His‐Ubq, and two recombinant proteins, His‐SHAN and His‐CS, expressed in
Escherichia coli Results Small proteins (His‐Ubq) and medium proteins (His‐SAHN) could readily be detected from 96‐well plates by direct infusion ESI, or from microscope slides by DESI‐MS after purification on surface from clarified
E. coli cell lysate. Protein oxidation was observed for immobilized proteins on both Cu‐NTA and Ni‐NTA; however, this did not hamper the enzymatic reactions of these proteins. Both the nucleosidase reaction products for His‐SAHN and the methylation product of His‐CS (theobromine to caffeine) were detected.Conclusions The immobilization, purification, release and detection of His‐tagged recombinant proteins using immobilized metal affinity surfaces for direct infusion ESI‐MS or ambient DESI‐MS analyses were successfully demonstrated. Recombinant proteins were purified to allow identification directly out of clarified cell lysate. Biological activities of the recombinant proteins were preserved allowing the investigation of enzymatic activity via MS.
-
more » « less
Abstract Nitroalkanes react specifically with aldehydes, providing rapid, stable, and chemoselective protein bioconjugation. These nitroalkylated proteins mimic key post‐translational modifications (PTMs) of proteins and can be used to understand the role of these PTMs in cellular processes. Demonstrated here is the substrate scope of this bioconjugation by attaching a variety of tags, such as NMR tags, fluorescent tags, affinity tags, and alkyne tags, to proteins. The structure and enzymatic activity of modified proteins remain conserved after labeling. Notably, the nitroalkane group leads to easy characterization of proteins by mass spectrometry because of its distinct fingerprint pattern. Importantly, the nitro‐alkylated peptides provide a new handle for site‐selective fluorination of peptides, thus installing a specific probe to study peptide–protein interactions by19F NMR spectroscopy. Furthermore, nitroalkane reagents can be used for the late‐stage diversification of peptides and for the synthesis of peptide staples.
