Efficient and Site-specific Antibody Labeling by Strain-promoted Azide-alkyne Cycloaddition

Summary

Here, we present a protocol to site-specifically introduce chemical probes into an antibody fragment by genetically incorporating an azide-containing amino acid, and subsequently coupling the azide with a chemical probe by strain-promoted azide-alkyne cycloaddition (SPAAC).

Abstract

There are currently many chemical tools available to introduce chemical probes into proteins to study their structure and function. A useful method is protein conjugation by genetically introducing an unnatural amino acid containing a bioorthogonal functional group. This report describes a detailed protocol for site-specific antibody conjugation. The protocol includes experimental details for the genetic incorporation of an azide-containing amino acid, and the conjugation reaction by strain-promoted azide-alkyne cycloaddition (SPAAC). This strain-promoted reaction proceeds by simple mixing of the reacting molecules at physiological pH and temperature, and does not require additional reagents such as copper(I) ions and copper-chelating ligands. Therefore, this method would be useful for general protein conjugation and development of antibody drug conjugates (ADCs).

Introduction

Since the genetic incorporation of p-methoxyphenylalanine in Escherichia coli was reported,¹ more than 100 unnatural amino acids (UAAs) have been successfully incorporated into various proteins.^1-3 Among these UAAs, the amino acids containing bioorthogonal functional groups have been extensively studied and represent the largest proportion. The bioorthogonal functional groups used in the UAAs include ketone,⁴ azide,⁵ alkyne,⁶ cyclooctyne,⁷ tetrazine,⁸ α,β-unsaturated amide,⁹ norbonene,¹⁰ transcyclooctene,¹¹ and bicyclo[6.1.0]-nonyne....

Protocol

1. Plasmid Construction

1. Construct an expression plasmid (pBAD-HerFab-L177TAG) that would express the target antibody gene (pBAD-HerFab-WT) with a His₆-tag, and replace the codon for Leucine-177 with the amber codon (TAG) ²⁷, using conventional site-directed mutagenesis technique.
   See Table of Materials.
2. Construct another expression plasmid (pEVOL-AFRS) containing the genes for the evolved tRNA^Tyr and aminoacyl-tRNA synthetase (aaRS) pair. Use the specially designed plasmid vector, pEVOL, for efficient incorporation of UAAs. The detailed plasmid information and cloning protocols are describe....

Results

In this study, an antibody fragment was site-specifically conjugated with a fluorophore by incorporating an azide-containing amino acid into the fragment and reacting the mutant antibody fragment with a strained cyclooctyne (Figure 1). HerFab was selected as the target antibody fragment into which AF was incorporated as an azide-containing amino acid. To choose the residue in HerFab for the replacement with AF, the X-ray crystal structure of HerFab was analyzed. ^{30 <.......}

Discussion

The genetic incorporation of unnatural amino acids into proteins has several advantages over other methods used for protein modification. ^1-3 One of the important advantages is its general applicability to any kind of protein. In principle, there is no limitation in selecting a target protein and a target site of the protein. However, replacement of a structurally or functionally important residue with a UAA may result in altering the structure and function of the target protein. Generally, residues that are e.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1. Plasmid Construction
plasmid pBAD_HerFab_L177TAG			Optionally contain the amber stop codon (TAG) at a desired position. Ko, W. et al. Efficient and Site-Specific Antibody Labeling by Strain-promoted Azide-Alkyne Cycloaddition. BKCS. 36 (9), 2352-2354, doi: 10.1002/bkcs.10423, (2015)
plasmid pEvol-AFRS			Young, T. S., Ahmad, I., Yin, J. A., and Schultz, P. G. An enhanced system for unnatural amino acid mutagenesis in E. coli. J. Mol. Biol. 395 (2), 361-374, doi: 10.1016/j.jmb.2009.10.030, (2010)
DH10B	Invitrogen	C6400-03	Expression Host
Plasmid Mini-prep kit	Nucleogen	5112	200/pack
Agarose	Intron biotechnology	32034	500 g
Ethidium bromide	Alfa Aesar	L07482	1 g
LB Broth	BD Difco	244620	500 g
2. Culture Preparation
2.1 Electroporation
Micro pulser	BIO-RAD	165-2100
Micro pulser cuvette	BIO-RAD	165-2089	0.1 cm electrode gap, pkg. of 50
Ampicillin Sodium	Wako	018-10372	25 g
Chloramphenicol	Alfa Aesar	B20841	25 g
Agar	SAMCHUN	214230	500 g
SOC medium	Sigma	S1797	100 mL
3. Expression and Purification of HerFab-L177AF
3.1 Expression of Herfab-L177AF
p-azido-L-phenylalanine (AF)	Bachem	F-3075.0001	1 g
L(+)-Arabinose, 99%	Acros	104981000	100 g
Hydrochloric acid, 35~37%	SAMCHUN	H0256	500 mL
3.2 Cell Lysis
Tris(hydroxymethyl)aminomethane, 99%	SAMCHUN	T1351	500 g
EDTA disodium salt dihydrate, 99.5%	SAMCHUN	E0064	1 kg
Sucrose	Sigma	S9378	500 g
Lysozyme	Siyaku	126-0671	1 g
3.3 Ni-NTA Affinity Chromatography
Ni-NTA resin	QIAGEN	30210	25 mL
Polypropylene column	QIAGEN	34924	50/pack, 1 mL capacity
Imidazole, 99%	SAMCHUN	I0578	1 kg
Sodium phosphate monobasic, 98%	SAMCHUN	S0919	1 kg
Sodium Chloride, 99%	SAMCHUN	S2907	1 kg
4. Conjugation of Purified HerFab-L177AF with Alkyne Probes Using Strain-promoted Azide-alkyne Cycloaddition (SPAAC)
Cy5.5-ADIBO	FutureChem	FC-6119	1 mg
5. Purification of Labeled HerFab
Amicon Ultra 0.5 mL Centrifugal Filters	MILLIPORE	UFC500396	96/pack, 500 μL capacity
6. SDS-PAGE Analysis of Labeled HerFab and Fluorescent Gel Scanning
1,4-Dithio-DL-threitol, DTT, 99.5%	Sigma	10708984001	10 g
NuPAGE LDS Sample Buffer, 4x	Thermofisher	NP0007	10 mL
MES running buffer	Thermofisher	NP0002	500 mL
Nupage Novex 4-12% SDS PAGE gels	Thermofisher	NO0321	12-well
Coomassie Brilliant Blue R-250	Wako	031-17922	25 g
Typhoon 9210 variable mode imager	Amersham Biosciences