Genetic Incorporation of Biosynthesized L-dihydroxyphenylalanine (DOPA) and Its Application to Protein Conjugation

Summary

Here, we present a protocol for the genetic incorporation of L-dihydroxyphenylalanine biosynthesized from simple starting materials and its application to protein conjugation.

Abstract

L-dihydroxyphenylalanine (DOPA) is an amino acid found in the biosynthesis of catecholamines in animals and plants. Because of its particular biochemical properties, the amino acid has multiple uses in biochemical applications. This report describes a protocol for the genetic incorporation of biosynthesized DOPA and its application to protein conjugation. DOPA is biosynthesized by a tyrosine phenol-lyase (TPL) from catechol, pyruvate, and ammonia, and the amino acid is directly incorporated into proteins by the genetic incorporation method using an evolved aminoacyl-tRNA and aminoacyl-tRNA synthetase pair. This direct incorporation system efficiently incorporates DOPA with little incorporation of other natural amino acids and with better protein yield than the previous genetic incorporation system for DOPA. Protein conjugation with DOPA-containing proteins is efficient and site-specific and shows its usefulness for various applications. This protocol provides protein scientists with detailed procedures for the efficient biosynthesis of mutant proteins containing DOPA at desired sites and their conjugation for industrial and pharmaceutical applications.

Introduction

DOPA is an amino acid involved in the biosynthesis of catecholamines in animals and plants. This amino acid is synthesized from Tyr by tyrosine hydroxylase and molecular oxygen (O₂)¹. Because DOPA is a precursor of dopamine and can permeate the blood-brain barrier, it has been used in the treatment of Parkinson's disease². DOPA is also found in mussel adhesion proteins (MAPs), which are responsible for the adhesive properties of mussels in wet conditions^3,4,5,6,7. Tyr is initially encoded at the positions where DOPA is found in MAPs and is then converted into DOPA by tyrosinases^8,9. Because of its interesting biochemical properties, DOPA has been used in a variety of applications. The dihydroxyl group of DOPA is chemically prone to oxidation, and the amino acid is easily converted into L-dopaquinone, a precursor of melanins. Owing to its high electrophilicity, L-dopaquinone and its derivatives have been used for crosslinking and conjugation with thiols and amines^10,11,12,13. 1,2-Quinones can also function as a diene for cycloaddition reactions and have been used for bioconjugation by strain-promoted oxidation-controlled cyclooctyne-1,2-quinone (SPOCQ) cycloaddition¹⁴. In addition, the dihydroxyl group can chelate metal ions such as Fe³⁺ and Cu²⁺, and proteins containing DOPA have been utilized for drug delivery and metal ion sensing^15,16.

DOPA has been genetically incorporated into proteins by using an orthogonal aminoacyl-tRNA (aa-tRNA) and aminoacyl-tRNA synthetase (aaRS) pair¹⁷ and used for protein conjugation and crosslinking^10,11,12,13. In this report, experimental results and protocols for the genetic incorporation of DOPA biosynthesized from cheap starting materials and its applications to bioconjugation are described. DOPA is biosynthesized using a TPL and starting from catechol, pyruvate, and ammonia in Escherichia coli. The biosynthesized DOPA is directly incorporated into proteins by expressing an evolved aa-tRNA and aaRS pair for DOPA. In addition, the biosynthesized protein containing DOPA is site-specifically conjugated with a fluorescent probe and crosslinked to produce protein oligomers. This protocol will be useful for protein scientists, to biosynthesize mutant proteins containing DOPA and conjugate the proteins with biochemical probes or drugs for industrial and pharmaceutical applications.

Protocol

1. Plasmid Construction

1. Construct an expression plasmid (pBAD-dual-TPL-GFP-WT) that expresses the TPL gene from Citrobacter freundii under the control of a constitutive promoter and the green fluorescent protein (GFP) gene with a His₆-tag under the control of the araBAD promoter. For pBAD-dual-TPL-GFP-E90TAG, replace the codon for the site (E90) of DOPA with an amber codon (TAG), using a site-directed mutagenesis protocol. The details for the construction of this plasmid was described in our previous report¹⁸.
2. Construct a tRNA/aaRS-expressing plasmid (pEVOL-DHPRS2)^18,19 for the genetic incorporation of DOPA. pEVOL is a special plasmid vector expressing two copies of aaRS genes with independent promoters, and the detailed information of the plasmid is described in a previous report¹⁹.
3. Use commercially available plasmid preparation kits to obtain high purity plasmid DNAs. Check the purity of the prepped DNAs by using agarose gel electrophoresis, if necessary.

2. Culture Preparation

1. Electroporation
   1. Use the electro-competent E. coli DH10β strain for this experiment. Add 1 µL of each plasmid DNA (pEVOL-DHPRS2 and pBAD-dual-TPL-GFP-E90TAG) to 20 µL of competent cells. Mix them gently to make a homogeneous mixture, using a pipette.
   2. Transfer the mixture to the electroporation cuvettes. Place the cuvette in the electroporation chamber. Push the cuvette into the chamber to make firm cuvette-chamber contact.
   3. Shock the cuvette, using 25 µF and 2.5 kV for 0.1-cm cuvettes.
   4. Add 1 mL of prewarmed super optimal broth (SOC), containing 2% (w/v) tryptone, 0.5% (w/v) yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, and 20 mM glucose, to the cuvette, and transfer the cells to a culture tube. Rescue the cells by incubating them for 1 h at 37 °C with shaking.
   5. Spread 10 - 100 µL of the rescued cells on a lysogeny broth (LB) agar plate containing 35 µg/mL chloramphenicol and 100 µg/mL ampicillin. Incubate the cells at 37 °C for 16 h.
2. Starter culture preparation
   1. Inoculate a single transformed colony in 5 mL of LB medium containing antibiotics. Incubate the colony for 12 h at 37 °C with shaking.

3. Expression and Purification of GFP-E90DOPA by a Biosynthetic System

1. Expression of GFP-E90DOPA by a biosynthetic system
   1. Transfer the starter culture (2 mL) to 100 mL of PA-5052 medium²⁰ (50 mM Na₂HPO₄, 50 mM KH₂PO₄, 25 mM [NH₄]₂SO₄, 2 mM MgSO₄, 0.1% [w/v] trace metals, 0.5% [w/v] glycerol, 0.05% [w/v] glucose, and 5% [w/v] amino acids [pH 7.25]) containing 35 µg/mL chloramphenicol, 100 µg/mL ampicillin, 100 mM pyruvate, 10 mM catechol, 25 mM ammonia, and 300 µM dithiothreitol (DTT) followed by an incubation at 30 °C with shaking.
   2. Add 2 mL of 0.2% (w/v) L-arabinose when the optical density (OD) at 550 nm reaches 0.8. Incubate the sample for 12 h at 30 °C with shaking.
   3. Spin down the cells at 11,000 x g for 5 min, discard the supernatant, and freeze the cell pellet at -80 °C.
2. Cell lysis
   1. Thaw the cell pellet on ice and resuspend the cells in 10 mL of lysis buffer containing 50 mM NaH₂PO₄ (pH 8.0), 10 mM imidazole, and 300 mM NaCl.
   2. Sonicate the resuspended cells on ice for 10 min. Use 80% sonication amplitude with a pulse of 25 s on and 35 s off.
   3. Spin down the cell lysate at 18,000 x g at 4 °C for 15 min. Transfer the supernatant to a fresh tube for purification and discard the pellet.
3. Ni-NTA affinity chromatography
   1. Add Ni-NTA (nitrilotriacetic acid) resin (300 µL of resin suspension for a 100-mL culture) to the supernatant and bind the proteins to the Ni-NTA resin at 4 °C by gently shaking the suspension for 1 h.
   2. Transfer the suspension to a polypropylene column and wash the resin 3x with 5 mL of wash buffer containing 50 mM NaH₂PO₄ (pH 8.0), 20 mM imidazole, and 300 mM NaCl.
   3. Elute the proteins 3x with 300 µL of elution buffer containing 50 mM NaH₂PO₄ (pH 8.0), 250 mM imidazole, and 300 mM NaCl.
   4. Determine the protein concentration by measuring the absorbance at 280 nm. The extinction coefficient (24,630 M^-1cm^-1) for GFP-E90DOPA at 280 nm was calculated by a protein extinction coefficient calculator (e.g., https://web.expasy.org/protparam/) and the independently measured extinction coefficiennt (2630 M^-1cm^-1) for DOPA. As an alternative method, use the Bradford protein assay²¹ for a determination of the protein concentration.

4. Oligomerization of Purified GFP-E90DOPA

1. Add 1 µL of sodium periodate in H₂O (6 mM) (1.0 equiv. to GFP-E90DOPA) to a solution of 20 µL of GFP-E90DOPA (300 µM) in a phosphate buffer containing 50 mM Na₂HPO₄ (pH 8.0) and 300 mM NaCl.
2. Allow the oligomerization reaction to proceed at 25 °C for 48 h.
3. Analyze the reaction mixture by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) (SDS-PAGE) as described in step 6.

5. Conjugation of GFP-E90DOPA with an Alkyne Probe by SPOCQ

1. Add 1 µL of the Cy5.5-linked azadibenzocyclooctyne (Cy5.5-ADIBO)²² in H₂O (4.0 mM) (10 equiv. to GFP-E90DOPA) to a solution of 20 µL of GFP-E90DOPA (20 µM) in a phosphate buffer containing 50 mM Na₂HPO₄ (pH 8.0) and 300 mM NaCl.
2. Add 1 µL of sodium periodate in H₂O (400 µM) (1.0 equiv. to GFP-E90DOPA) to the reaction mixture.
3. Allow the SPOCQ reaction to proceed at 25 °C for 1 h. If a light-sensitive probe is used, cover the reaction vessel with aluminum foil.

6. Purification of the Labeled GFP

1. Dilute the SPOCQ reaction mixture by adding a phosphate buffer, containing 50 mM Na₂HPO₄ (pH 8.0) and 300 mM NaCl, up to 500 µL.
2. Transfer the diluted solution to a centrifugal filter spin column. Centrifuge the spin column at 14,000 x g at 4 °C for 15 min and discard the flow-through. Repeat this step 3x in order to remove any excess Cy5.5-ADIBO.
3. Transfer the purified sample from the spin column to a 1.5-mL microcentrifuge tube.
4. Alternatively, carry out a purification by dialysis against the same buffer.
5. Store the purified GFP at 4 °C.

7. SDS-PAGE Analysis and Fluorescence Gel Scanning

1. Prepare protein samples for SDS-PAGE analysis by adding 5 µL of protein sample buffer (see Table of Materials) and 2 µL of DTT (1.00 M) to 13 µL of purified, or crude, labeled GFP (13.6 µM or 0.38 mg/mL). For an analysis of oligomeric GFP, use a higher concentration of GFP (67.8 µM or 1.9 mg/mL). Denature the proteins by incubating them at 95 °C for 10 min.
2. Place a 4% - 12% Bis-Tris SDS-PAGE gel cassette (purchased, premade gel cassettes) to the electrophoresis cell and add a running buffer (see Table of Materials). Load the protein samples and a molecular weight marker. Run the electrophoresis for 35 - 40 min at 200 V.
   NOTE: Avoid any exposure of the protein samples to light by carrying out all processes in the dark, to minimize photo-bleaching of the fluorescent probe.
3. Use a fluorescence scanner for fluorescence imaging. Wrap a protein gel from electrophoresis and place it on a fluorescence scanner. Scan the gel by using an appropriate wavelength setting. For Cy5.5, select the Cy5 mode provided in the scanner software.
4. Stain the gel with a commercial protein staining reagent.

8. MALDI-TOF MS Analysis by Trypsin Digestion

1. Incubate 100 µL of purified GFP-E90DOPA (2.2 mg/mL or 80 µM) in a buffer containing 50 mM Tris-HCl (pH 8.0), 0.1% (w/v) SDS, and 25 mM DTT at 60 °C for 1 h.
2. Add 1 µL of Iodoacetamide in H₂O (IAA, 4.0 M) (500 equiv. to GFP-E90DOPA) to the purified GFP-E90DOPA solution (80 µM) in the same buffer. Incubate the mixture at 25 °C for 30 min with protection from light.
3. Digest the GFP-E90DOPA sample by adding trypsin (1:100 protease-to-substrate-protein ratio [w/w]) and incubate the reaction mixture at 37 °C for 4 h.
4. Purify the digested peptide sample by using a standard desalting method with C₁₈ spin columns.
5. Analyze the digested peptides by the reported protocol for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) using alpha-cyano-4-hydroxycinnamic acid (CHCA) as a matrix²³.

Results

The expression system for the direct incorporation of DOPA biosynthesized from a TPL is shown in Figure 1. The genes for the evolved aa-tRNA and aaRS pair are placed in a plasmid, and the GFP gene (GFP-E90TAG) containing an amber codon at position 90 is located in another plasmid to evaluate the incorporation of DOPA by GFP fluorescence. The TPL gene is placed in the same expression plasmid containing the GFP gene and constitutively expressed to maximize the ...

Discussion

In this protocol, the biosynthesis and direct incorporation of DOPA are described. The bacterial cell used in this method can synthesize an additional amino acid and use it as an unnatural building block for protein synthesis. The genetic incorporation of unnatural amino acids has been a key technology for the development of unnatural organism with an expanded genetic code. However, this method has been technically incomplete and is being modified to improve incorporation efficiency and minimize perturbation to endogenou...

Materials

Name	Company	Catalog Number	Comments
1. Plasmid Construction
Plasmid pBAD-dual-TPL-GFP-E90TAG			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-DHPRS2			1. 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) 2. Kim, S., Sung, B. H., Kim, S. C., Lee, H. S. Genetic incorporation of l-dihydroxyphenylalanine (DOPA) biosynthesized by a tyrosine phenol-lyase. Chem. Commun. 54 (24), 3002-3005, doi: 10.1039/c8cc00281a (2018).
DH10β	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 GFP-E90DOPA by biosynthetic system
3.1 Expression of GFP-E90DOPA by biosynthetic system
L(+)-Arabinose, 99%	Acros	104981000	100 g
Pyrocatechol, 99%	SAMCHUN	P1387	25 g
Ammonium sulfate, 99%	SAMCHUN	A0943	500 g
pyruvic acid, 98%	Alfa Aesar	A13875	100 g
Sodium phosphate dibasic, anhydrous, 99%	SAMCHUN	S0891	1 kg
Potassium phophate, monobasic, 99%	SAMCHUN	P1127	1 kg
Magnesium sulfate, anhydrous, 99%	SAMCHUN	M0146	1 kg
D(+)-Glucose, anhydrous, 99%	SAMCHUN	D0092	500 g
Glycerol, 99%	SAMCHUN	G0269	1 kg
Trace metal mix a5 with co	Sigma	92949	25 mL
L-Proline, 99%	SAMCHUN	P1257	25 g
L-Phenylalanine, 98.5%	SAMCHUN	P1982	25 g
L-Tryptophane	JUNSEI	49550-0310	25 g
L-Arginine, 98%	SAMCHUN	A1149	25 g
L-Glutamine, 98%	JUNSEI	27340-0310	25 g
L-Asparagine monohydrate, 99%	SAMCHUN	A1198	25 g
L-Methionine	JUNSEI	73190-0410	25 g
L-Histidine hydrochloride monohydrate, 99%	SAMCHUN	H0604	25 g
L-Threonine, 99%	SAMCHUN	T2938	25 g
L-Leucine	JUNSEI	87070-0310	25 g
Glycine, 99%	SAMCHUN	G0286	25 g
L-Glutamic acid, 99%	SAMCHUN	G0233	25 g
L-Alanine, 99%	SAMCHUN	A1543	25 g
L-Isoleucine, 99%	SAMCHUN	I1049	25 g
L-Valine, 99%	SAMCHUN	V0088	25 g
L-Serine	SAMCHUN	S2447	25 g
L-Aspartic acid	SAMCHUN	A1205	25 g
L-Lysine monohydrochloride, 99%	SAMCHUN	L0592	25 g
3.2 Cell lysis
Imidazole, 99%	SAMCHUN	I0578	1kg
Sodium phosphate monobasic, 98%	SAMCHUN	S0919	1 kg
Sodium Chloride, 99%	SAMCHUN	S2907	1 kg
Ultrasonic Processor - 150 microliters to 150 milliliters	SONIC & MATERIALS	VCX130
3.3 Ni-NTA Affinity Chromatography
Ni-NTA resin	QIAGEN	30210	25 mL
Polypropylene column	QIAGEN	34924	50/pack, 1 mL capacity
4. Oligomerization of Purified GFP-E90DOPA
Sodium periodate, 99.8&	Acros	419610050	5 g
5. Conjugation of GFP-E90DOPA with an Alkyne Probe by Strain-Promoted Oxidation-Controlled Cyclooctyne–1,2-Quinone Cycloaddition (SPOCQ)
Cy5.5-ADIBO	FutureChem	FC-6119	1mg
6. Purification of Labeled GFP
Amicon Ultra 0.5 mL Centrifugal Filters	MILLIPORE	UFC500396	96/pack, 500ul capacity
7. SDS-PAGE Analysis and Fluorescence 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
G:BOX Chemi Fluorescent & Chemiluminescent Imaging System	Syngene		G BOX Chemi XT4
8. MALDI-TOF MS analysis by Trypsin Digestion
8.1 Preparation of the digested peptide sample by trypsin digestion
Tris(hydroxymethyl)aminomethane, 99%	SAMCHUN	T1351	500 g
Hydrochloric acid, 35~37%	SAMCHUN	H0256	500 mL
Dodecyl sulfate sodium salt, 85%	SAMCHUN	D1070	250 g
Iodoacetamide	Sigma	I6125	5 g
Trypsin Protease, MS Grade	Thermofisher	90057	5 x 20 µg/pack
C-18 spin columns	Thermofisher	89870	25/pack, 200 µL capacity
8.2 Analysis of the digested peptide by MALDI-TOF
Acetonitirile, 99.5%	SAMCHUN	A0125	500 mL
α-Cyano-4-hydroxycinnamic acid	Sigma	C2020	10 g
Trifluoroacetic acid, 99%	SAMCHUN	T1666	100 g
MTP 384 target plate ground steel BC targets	Bruker	8280784
Bruker Autoflex Speed MALDI-TOF mass spectrometer	Bruker