This research was supported by the Global Frontier Research Program (NRF-2015M3A6A8065833), and the Basic Science&#160;Research Program (2018R1A6A1A03024940)&#160;through the National Research Foundation of Korea (NRF) funded&#160;by the Korea government.&#160;

1. Plasmid Construction
<ol>
	<li>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 o...

<ol>
	<li>Nagatsu, T., Levitt, M., Udenfriend, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tyrosine+hydroxylase:+The+initial+step+in+norepinephrine+biosynthesis.">Tyrosine hydroxylase: The initial step in norepinephrine biosynthesis.</a> Journal of Biological Chemistry. 239, 2910-2917 (1964).</li><li>Pinder, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Possible+dopamine+derivatives+capable+of+crossing+the+blood-brain+barrier+in+relation+to+parkinsonism.">Possible dopamine derivatives capable of crossing the blood-brain barrier in relation to parkinsonism.</a> Nature. 228 (5269), 358 (1970).</li><li>Waite, J. H., Tanzer, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyphenolic+substance+of+mytilus+edulis:+novel+adhesive+containing+L-DOPA+and+hydroxyproline.">Polyphenolic substance of mytilus edulis: novel adhesive containing L-DOPA and hydroxyproline.</a> Science. 212 (4498), 1038-1040 (1981).</li><li>Lee, H., Scherer, N. F., Messersmith, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+mechanics+of+mussel+adhesion.">Single-molecule mechanics of mussel adhesion.</a> Proceedings of the National Academy of Sciences of the United States of America. 103 (35), 12999-13003 (2006).</li><li>Papov, V. V., Diamond, T. V., Biemann, K., Waite, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydroxyarginine-containing+polyphenolic+proteins+in+the+adhesive+plaques+of+the+marine+mussel+Mytilus+edulis.">Hydroxyarginine-containing polyphenolic proteins in the adhesive plaques of the marine mussel Mytilus edulis.</a> Journal of Biological Chemistry. 270 (34), 20183-20192 (1995).</li><li>Waite, J. H., Qin, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyphosphoprotein+from+the+adhesive+pads+of+Mytilus+edulis.">Polyphosphoprotein from the adhesive pads of Mytilus edulis.</a> Biochemistry. 40 (9), 2887-2893 (2001).</li><li>Nicklisch, S. C., Waite, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mini-review:+The+role+of+redox+in+DOPA-mediated+marine+adhesion.">Mini-review: The role of redox in DOPA-mediated marine adhesion.</a> Biofouling. 28 (8), 865-877 (2012).</li><li>Silverman, H. G., Roberto, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+marine+mussel+adhesion.">Understanding marine mussel adhesion.</a> Marine Biotechnology. 9 (6), 661-681 (2007).</li><li>Lee, B. P., Messersmith, P. B., Israelachvili, J. N., Waite, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mussel-inspired+adhesives+and+coatings.">Mussel-inspired adhesives and coatings.</a> Annual Review of Materials Research. 41, 99-132 (2011).</li><li>Umeda, A., Thibodeux, G. N., Zhu, J., Lee, Y., Zhang, Z. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+protein+cross-linking+with+genetically+incorporated+3,4-dihydroxy-L-phenylalanine.">Site-specific protein cross-linking with genetically incorporated 3,4-dihydroxy-L-phenylalanine.</a> ChemBioChem. 10 (8), 1302-1304 (2009).</li><li>Umeda, A., Thibodeux, G. N., Moncivais, K., Jiang, F., Zhang, Z. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+versatile+approach+to+transform+low-affinity+peptides+into+protein+probes+with+cotranslationally+expressed+chemical+cross-linker.">A versatile approach to transform low-affinity peptides into protein probes with cotranslationally expressed chemical cross-linker.</a> Analytical Biochemistry. 405 (1), 82-88 (2010).</li><li>Xu, J., Tack, D., Hughes, R. A., Ellington, A. D., Gray, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-based+non-canonical+amino+acid+design+to+covalently+crosslink+an+antibody-antigen+complex.">Structure-based non-canonical amino acid design to covalently crosslink an antibody-antigen complex.</a> Journal of Structural Biology. 185 (2), 215-222 (2014).</li><li>Burdine, L., Gillette, T. G., Lin, H. J., Kodadek, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Periodate-triggered+cross-linking+of+DOPA-containing+peptide-protein+complexes.">Periodate-triggered cross-linking of DOPA-containing peptide-protein complexes.</a> Journal of the American Chemical Society. 126 (37), 11442-11443 (2004).</li><li>Borrmann, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain-promoted+oxidation-controlled+cyclooctyne-1,2-quinone+cycloaddition+(SPOCQ)+for+fast+and+activatable+protein+conjugation.">Strain-promoted oxidation-controlled cyclooctyne-1,2-quinone cycloaddition (SPOCQ) for fast and activatable protein conjugation.</a> Bioconjugate Chemistry. 26 (2), 257-261 (2015).</li><li>Ayyadurai, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+selective,+sensitive,+and+reversible+biosensor+by+the+genetic+incorporation+of+a+metal-binding+site+into+green+fluorescent+protein.">Development of a selective, sensitive, and reversible biosensor by the genetic incorporation of a metal-binding site into green fluorescent protein.</a> Angewandte Chemie International Edition. 50 (29), 6534-6537 (2011).</li><li>Kim, B. J., Cheong, H., Hwang, B. H., Cha, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mussel-inspired+protein+nanoparticles+containing+iron(III)-DOPA+complexes+for+pH-responsive+drug+delivery.">Mussel-inspired protein nanoparticles containing iron(III)-DOPA complexes for pH-responsive drug delivery.</a> Angewandte Chemie International Edition. 54 (25), 7318-7322 (2015).</li><li>Alfonta, L., Zhang, Z., Uryu, S., Loo, J. A., Schultz, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+incorporation+of+a+redox-active+amino+acid+into+proteins.">Site-specific incorporation of a redox-active amino acid into proteins.</a> Journal of the American Chemical Society. 125 (48), 14662-14663 (2003).</li><li>Kim, S., Sung, B. H., Kim, S. C., Lee, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+incorporation+of+L-dihydroxyphenylalanine+(DOPA)+biosynthesized+by+a+tyrosine+phenol-lyase.">Genetic incorporation of L-dihydroxyphenylalanine (DOPA) biosynthesized by a tyrosine phenol-lyase.</a> Chemical Communications. 54 (24), 3002-3005 (2018).</li><li>Young, T. S., Ahmad, I., Yin, J. A., Schultz, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+enhanced+system+for+unnatural+amino+acid+mutagenesis+in+E.+coli.">An enhanced system for unnatural amino acid mutagenesis in E. coli.</a> Journal of Molecular Biology. 395 (2), 361-374 (2010).</li><li>Studier, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+production+by+auto-induction+in+high+density+shaking+cultures.">Protein production by auto-induction in high density shaking cultures.</a> Protein Expression and Purification. 41 (1), 207-234 (2005).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Analytical Biochemistry. 72 (1-2), 248-254 (1976).</li><li>Jang, S., Sachin, K., Lee, H. J., Kim, D. W., Lee, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+simple+method+for+protein+conjugation+by+copper-free+click+reaction+and+its+application+to+antibody-free+Western+blot+analysis.">Development of a simple method for protein conjugation by copper-free click reaction and its application to antibody-free Western blot analysis.</a> Bioconjugate Chemistry. 23 (11), 2256-2261 (2012).</li><li>Gobom, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha-cyano-4-hydroxycinnamic+acid+affinity+sample+preparation.+A+protocol+for+MALDI-MS+peptide+analysis+in+proteomics.">Alpha-cyano-4-hydroxycinnamic acid affinity sample preparation. A protocol for MALDI-MS peptide analysis in proteomics.</a> Analytical Chemistry. 73 (3), 434-438 (2001).</li><li>Mukai, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+reproductive+Escherichia+coli+cells+with+no+specific+assignment+to+the+UAG+codon.">Highly reproductive Escherichia coli cells with no specific assignment to the UAG codon.</a> Scientific Reports. 5, 9699 (2015).</li><li>Lajoie, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomically+recoded+organisms+expand+biological+functions.">Genomically recoded organisms expand biological functions.</a> Science. 342 (6156), 357-360 (2013).</li><li>Bryson, D. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+directed+evolution+of+aminoacyl-tRNA+synthetases.">Continuous directed evolution of aminoacyl-tRNA synthetases.</a> Nature Chemical Biology. 13 (12), 1253-1260 (2017).</li><li>Guo, J., Melancon, C. E., Lee, H. S., Groff, D., Schultz, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+amber+suppressor+tRNAs+for+efficient+bacterial+production+of+proteins+containing+nonnatural+amino+acids.">Evolution of amber suppressor tRNAs for efficient bacterial production of proteins containing nonnatural amino acids.</a> Angewandte Chemie International Edition. 48 (48), 9148-9151 (2009).</li><li>Lee, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+facile+strategy+for+selective+phosphoserine+incorporation+in+histones.">A facile strategy for selective phosphoserine incorporation in histones.</a> Angewandte Chemie International Edition. 52 (22), 5771-5775 (2013).</li><li>Neumann, H., Wang, K., Davis, L., Garcia-Alai, M., Chin, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encoding+multiple+unnatural+amino+acids+via+evolution+of+a+quadruplet-decoding+ribosome.">Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome.</a> Nature. 464 (7287), 441-444 (2010).</li><li>Lang, K., Chin, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioorthogonal+reactions+for+labeling+proteins.">Bioorthogonal reactions for labeling proteins.</a> ACS Chemical Biology. 9 (1), 16-20 (2014).</li><li>Lang, K., Chin, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+Incorporation+of+unnatural+amino+acids+and+bioorthogonal+labeling+of+proteins.">Cellular Incorporation of unnatural amino acids and bioorthogonal labeling of proteins.</a> Chemical Reviews. 114 (9), 4764-4806 (2014).</li><li>Plass, T., Milles, S., Koehler, C., Schultz, C., Lemke, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoded+copper-free+click+chemistry.">Genetically encoded copper-free click chemistry.</a> Angewandte Chemie International Edition. 50 (17), 3878-3881 (2011).</li><li>Seitchik, J. 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The authors have nothing to disclose.

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...

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&#39;s disease2. DOPA is also found in mussel adhesion proteins (MAPs), which are responsible for the adhesive properties of mussels in wet conditions3,4,5,6,7. Tyr is initially encoded at the positions where DOPA is found in MAPs and is then converted into DOPA by tyrosinases8,9. 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 amines10,11,12,13. 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 sensing15,16.
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 crosslinking10,11,12,13. 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.

<table border="0" cellpadding="0" cellspacing="0" style="width:1235px;" width="1234">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">1. Plasmid Construction</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:596px;">Plasmid pBAD-dual-TPL-GFP-E90TAG</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td>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)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Plasmid pEvol-DHPRS2</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td>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).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">DH10&#946;</td>
			<td style="width:167px;">Invitrogen</td>
			<td style="width:133px;">C6400-03</td>
			<td>Expression Host</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Plasmid Mini-prep kit</td>
			<td style="width:167px;">Nucleogen</td>
			<td style="width:133px;">5112</td>
			<td>200/pack</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Agarose</td>
			<td style="width:167px;">Intron biotechnology</td>
			<td style="width:133px;">32034</td>
			<td>500 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Ethidium bromide</td>
			<td style="width:167px;">Alfa Aesar</td>
			<td style="width:133px;">L07482</td>
			<td>1 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">LB Broth</td>
			<td style="width:167px;">BD Difco</td>
			<td style="width:133px;">244620</td>
			<td>500 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:596px;">2. Culture preparation</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:596px;">2.1) Electroporation</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Micro pulser&#160;</td>
			<td style="width:167px;">BIO-RAD</td>
			<td style="width:133px;">165-2100</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Micro pulser cuvette</td>
			<td style="width:167px;">BIO-RAD</td>
			<td style="width:133px;">165-2089</td>
			<td style="width:339px;">0.1 cm electrode gap, pkg. of 50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Ampicillin Sodium</td>
			<td style="width:167px;">Wako</td>
			<td style="width:133px;">018-10372</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Chloramphenicol</td>
			<td style="width:167px;">Alfa Aesar</td>
			<td style="width:133px;">B20841</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Agar</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">214230</td>
			<td>500 g</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:596px;">SOC medium</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:133px;">S1797</td>
			<td>100 mL</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:596px;">3. Expression and Purification of GFP-E90DOPA by biosynthetic system</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:596px;">3.1 Expression of GFP-E90DOPA by biosynthetic system</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">L(+)-Arabinose, 99%</td>
			<td style="width:167px;">Acros</td>
			<td style="width:133px;">104981000</td>
			<td>100 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Pyrocatechol, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">P1387</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Ammonium sulfate, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">A0943</td>
			<td>500 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">pyruvic acid, 98%</td>
			<td style="width:167px;">Alfa Aesar</td>
			<td style="width:133px;">A13875</td>
			<td>100 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:596px;">Sodium phosphate dibasic, anhydrous, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">S0891</td>
			<td>1 kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Potassium phophate, monobasic, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">P1127</td>
			<td>1 kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Magnesium sulfate, anhydrous, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">M0146</td>
			<td>1 kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">D(+)-Glucose, anhydrous, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">D0092</td>
			<td>500 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Glycerol, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">G0269</td>
			<td>1 kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Trace metal mix a5 with co</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:133px;">92949</td>
			<td>25 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">L-Proline, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">P1257</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">L-Phenylalanine, 98.5%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">P1982</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">L-Tryptophane</td>
			<td style="width:167px;">JUNSEI</td>
			<td style="width:133px;">49550-0310</td>
			<td>25 g</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:596px;">L-Arginine, 98%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">A1149</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">L-Glutamine, 98%</td>
			<td style="width:167px;">JUNSEI</td>
			<td style="width:133px;">27340-0310</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">L-Asparagine monohydrate, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">A1198</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">L-Methionine</td>
			<td style="width:167px;">JUNSEI</td>
			<td style="width:133px;">73190-0410</td>
			<td>25 g</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:596px;">L-Histidine hydrochloride monohydrate, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">H0604</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">L-Threonine, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">T2938</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">L-Leucine</td>
			<td style="width:167px;">JUNSEI</td>
			<td style="width:133px;">87070-0310</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Glycine, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">G0286</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">L-Glutamic acid, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">G0233</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">L-Alanine, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">A1543</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">L-Isoleucine, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">I1049</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">L-Valine, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">V0088</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">L-Serine</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">S2447</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">L-Aspartic acid</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">A1205</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">L-Lysine monohydrochloride, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">L0592</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">3.2 Cell lysis</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Imidazole, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">I0578</td>
			<td>1kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Sodium phosphate monobasic, 98%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">S0919</td>
			<td>1 kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Sodium Chloride, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">S2907</td>
			<td>1 kg</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:596px;">Ultrasonic Processor - 150 microliters to 150 milliliters</td>
			<td style="width:167px;">SONIC &#38; MATERIALS</td>
			<td style="width:133px;">VCX130</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">3.3 Ni-NTA Affinity Chromatography</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Ni-NTA resin</td>
			<td style="width:167px;">QIAGEN</td>
			<td style="width:133px;">30210</td>
			<td>25 mL</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:596px;">Polypropylene column</td>
			<td style="width:167px;">QIAGEN</td>
			<td style="width:133px;">34924</td>
			<td>50/pack, 1 mL capacity</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:596px;">4. Oligomerization of Purified GFP-E90DOPA&#160;</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:596px;">Sodium periodate, 99.8&#38;</td>
			<td style="width:167px;">Acros</td>
			<td style="width:133px;">419610050</td>
			<td>5 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:596px;">5. Conjugation of GFP-E90DOPA with an Alkyne Probe by Strain-Promoted Oxidation-Controlled Cyclooctyne&#8211;1,2-Quinone Cycloaddition (SPOCQ)&#160;</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:596px;">Cy5.5-ADIBO&#160;</td>
			<td style="width:167px;">FutureChem</td>
			<td style="width:133px;">FC-6119</td>
			<td>1mg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">6. Purification of Labeled GFP</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:596px;">Amicon Ultra 0.5 mL Centrifugal Filters</td>
			<td style="width:167px;">MILLIPORE</td>
			<td style="width:133px;">UFC500396</td>
			<td>96/pack, 500ul capacity</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:596px;">7. SDS-PAGE Analysis and Fluorescence Gel Scanning</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">1,4-Dithio-DL-threitol, DTT, 99.5 %</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:133px;">10708984001</td>
			<td>10 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">NuPAGE LDS Sample Buffer, 4X</td>
			<td style="width:167px;">Thermofisher</td>
			<td style="width:133px;">NP0007</td>
			<td>10 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">MES running buffer</td>
			<td style="width:167px;">Thermofisher</td>
			<td style="width:133px;">NP0002</td>
			<td>500 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Nupage Novex 4-12% SDS PAGE gels</td>
			<td style="width:167px;">Thermofisher</td>
			<td style="width:133px;">NO0321</td>
			<td>12 well</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Coomassie Brilliant Blue R-250</td>
			<td style="width:167px;">Wako</td>
			<td style="width:133px;">031-17922</td>
			<td>25 g</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:596px;">G:BOX Chemi Fluorescent &#38; Chemiluminescent Imaging System</td>
			<td style="width:167px;">Syngene</td>
			<td style="width:133px;"></td>
			<td>G BOX Chemi XT4</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:596px;">8. MALDI-TOF MS analysis by Trypsin Digestion</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:596px;">8.1 Preparation of the digested peptide sample by trypsin digestion</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Tris(hydroxymethyl)aminomethane, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">T1351</td>
			<td>500 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Hydrochloric acid, 35~37%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">H0256</td>
			<td>500 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Dodecyl sulfate sodium salt, 85%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">D1070</td>
			<td>250 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Iodoacetamide</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:133px;">I6125</td>
			<td>5 g</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:596px;">Trypsin Protease, MS Grade</td>
			<td style="width:167px;">Thermofisher</td>
			<td style="width:133px;">90057</td>
			<td>5 x 20 &#181;g/pack</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">C-18 spin columns</td>
			<td style="width:167px;">Thermofisher</td>
			<td style="width:133px;">89870</td>
			<td>25/pack, 200 &#181;L capacity</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:596px;">8.2 Analysis of the digested peptide by MALDI-TOF</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Acetonitirile, 99.5%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">A0125</td>
			<td>500 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">&#945;-Cyano-4-hydroxycinnamic acid</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:133px;">C2020</td>
			<td>10 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Trifluoroacetic acid, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">T1666</td>
			<td>100 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">MTP 384 target plate ground steel BC targets</td>
			<td style="width:167px;">Bruker</td>
			<td style="width:133px;">8280784</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Bruker Autoflex Speed MALDI-TOF mass spectrometer</td>
			<td style="width:167px;">Bruker</td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

genetic incorporation of biosynthesized l-dihydroxyphenylalanine (dopa) and its application to protein conjugation

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.

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 ...

Watch this Scientific Journal Video about Genetic Incorporation of Biosynthesized L-dihydroxyphenylalanine DOPA and Its Application to Protein Conjugation at JoVE.com

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

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

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

L-dihydroxyphenylalanine (DOPA) is an amino acid found in the biosynthesis of catecholamines in animals and plants. Because of its particular biochemical ...

This method can help to answer key questions in the chemical biology field, such as protein modification, conjugation, and biosynthetic system. The main advantage of this technique is that L-DOPA can be cited specifically and efficiently introducing the Tyr proteins by using biosynthetic system. After constructing the expression plasmid, set out electrocompetent E.coli DH10 beta strain cells for this procedure.Add one microliter of each plasmid DNA to 20 microliters of competent cells. Use a pipette to gently mix. Transfer this mixture to 0.1 centimeter electroporation cuvettes.Place a filled cuvette in the electroporation chamber, making sure there is firm cuvette chamber contact. Shock the cuvette at 25 microfarad and 2.4 kilovolts. Then, add one milliliter of pre-warmed SOC medium.Transfer the cells to a culture tube. Incubate at 37 degrees Celsius for one hour with shaking to rescue the cells. Spread 10 to 100 microliters of the rescued cells on a lysogeny broth agar plate that contains 35 micrograms per milliliter chloramphenicol and 100 micrograms per milliliter ampicillin.Incubate at 37 degrees Celsius for 16 hours. After this, inoculate a single transformed colony in five milliliters of LB medium containing antibiotics to prepare a starter culture. Incubate at 37 degrees Celsius with shaking for 12 hours.Transfer two milliliters of the starter culture into 100 milliliters of PA-5052 medium, containing chloramphenicol, ampicillin, pyruvate, catechol, ammonia, and dithiothreitol. Incubate at 30 degrees celsius with shaking. When the optical density at 550 nanometers reaches 0.8, add one milliliter of 20%L-arabinose.Incubate the sample at 30 degrees Celsius with shaking for 12 hours. After this, spin down the cells at 11000 times gravity for five minutes. Discard the supernatant and freeze the cell pellet at 80 degrees Celsius until ready to lyse the cells.When ready, thaw the cell pellet on ice. Resuspend the thawed cells in 10 milliliters of lysis buffer containing monosodium phosphate, imidazole, and sodium chloride. Use an 80%sonication amplitude with a pulse of 25 seconds off and 35 seconds off to sonicate the resuspended cells on ice for 10 minutes.Next, spin down the cell lysate at 18000 times gravity at four degrees Celsius for 15 minutes. Transfer the supernatant to a fresh tube for purification and discard the pellet. For a 100 milliliter culture, add 300 microliters of nickel-NTA resin suspension to the collected supernatant.Gently shake the suspension for one hour at four degrees Celsius to bind the proteins to the resin. After this, transfer the suspension to a polypropylene column. Wash the resin three times, using five milliliters of wash buffer for each wash.Then, use 300 microliters of elusion buffer to elute the proteins three times. Determine the protein concentration as outlined in the text protocol. To begin, prepare a 20 microliter solution containing 300 micromolar GFP containing DOPA at position 90 in a phosphate buffer containing 50 millimolar disodium phosphate and 300 millimolar sodium chloride.Add one microliter of a solution containing six millimolar of sodium periodate in water. Let the oligomerization reaction proceed at 25 degrees Celsius for 48 hours. After this, add five microliters of protein sample buffer and two microliters of dithiothreitol to 13 microliters of the oligomerization of GFP in order to prepare the protein samples for SDS page analysis.Incubate at 95 degrees Celsius for 10 minutes to denature the proteins. Next, place a 4%to 12%Bis-Tris SDS page gel cassette in the electrophoresis cell. Add a running buffer to the cassette.Load the protein samples and a molecular weight marker. Run the electrophoresis for 35 to 40 minutes at 200 volts. After this, stain the gel with a commercial protein staining reagent.First, prepare a 20 micromolar solution of GFP containing DOPA at position 90, and a phosphate buffer containing 50 millimolar disodium phosphate and 300 millimolar sodium chloride. Add one microliter of the Cy5.5 linked acid dibenzocyclooctyne. Next, add one microliter of a solution containing 400 micromolar sodium periodate in water to the reaction mixture.Let the strain promoted oxidation controlled cyclooctyne 1, 2-quinone cycloaddition reaction proceed at 25 degrees Celsius for one hour. If a light sensitive probe is used, cover the reaction vessel with aluminum foil. To begin purifying the labeled GFP, dilute the SPOCQ reaction mixture with a phosphate buffer containing 50 millimolar disodium phosphate and 300 millimolar sodium chloride up to 500 microliters.Transfer the diluted solution to a centrifugal filter spin column. Centrifuge the spin column at 14000 times gravity at four degrees Celsius for 15 minutes and discard the flowthrough. First, add five microliters of protein sample buffer and two microliters of dithiothreitol to 13 microliters of purified and labeled GFP in order to prepare the protein samples for SDS-PAGE analysis.Incubate at 95 degrees Celsius for 10 minutes to denature the proteins. Next, place a 4%to 12%Bis-Tris SDS page gel cassette in the electrophoresis cell. Add a running buffer to the cassette.Load the protein samples and a molecular weight marker. Run the electrophoresis for 35 to 40 minutes at 200 volts in the dark. After this, wrap a protein gel and place it on the fluorescence scanner.Use an appropriate wavelength setting to scan the gel. For Cy5.5, select the Cy5 mode provided in the scanning software. Then, stain the gel with a commercial protein staining reagent.In this study, the optimal conditions for DOPA biosynthesis are screened. And the biosynthesized DOPA is directly incorporated into GFP. The expressed mutant, GFP containing DOPA at position 90, is purified and analyzed by SDS-PAGE.The full length GFP is then purified and the DOPA incorporation was confirmed by MALDI-TOF MS analysis. While the incorporation efficiency of biosynthesized DOPA is seen to be similar to the general genetic incorporation efficiency of 3 millimolar DOPA, the cell density after 16 hours of culture is three to fourfold higher. The purified protein yield is also seen to be threefold higher from biosynthesis when compared to the general genetic incorporation.GFP containing DOPA at position 90 is then tested for SPOCQ cycloaddition. An intense fluorescence band is observed when treated with sodium periodate and Cy5.5 ADIBO, while no fluorescence band is seen in control reactions. This confirms the efficiency and specificity of the conjugation reaction with the genetically incorporated DOPA.Lastly, DOPA containing GFP is used for protein oligomerization. A clear oligomerization pattern can be seen with equally spaced protein bands for the periodate treated sample, while the control sample showed the intact protein without any other bands. After its development, this technique paved the way for researchers in the field of chemical biology to explore biochemical applications by L-DOPA containing proteins in a designed model organism.

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JoVE Journal

Biochemistry

7.8K vues.  Sogang University. This method can help to answer key questions in the chemical biology field, such as protein modification, conjugation, and biosynthetic system. The main advantage of this technique is that L-DOPA can be cited specifically and efficiently introducing the Tyr proteins by using biosynthetic system. After constructing the expression plasmid, set out electrocompetent E.coli DH10 beta strain cells for this procedure.Add one microliter of each plasmid DNA to 20 microliters of competent cells....