Genetiske indarbejdelse af Biosynthesized L-dihydroxyphenylalanine (DOPA) og dens anvendelse på Protein konjugation
Summary
Vi præsenterer her, en protokol for den genetiske indarbejdelse af L-dihydroxyphenylalanine biosynthesized fra simple udgangsmaterialer og dens anvendelse på protein konjugering.
Abstract
L-dihydroxyphenylalanine (DOPA) er en aminosyre, der findes i biosyntesen af katekolaminer i dyr og planter. På grund af dens særlige biokemiske egenskaber har aminosyren flere anvendelser i biokemiske anvendelser. Denne rapport beskriver en protokol til den genetiske indbygning af biosynthesized DOPA og dens anvendelse på protein konjugering. DOPA er biosynthesized af en tyrosin phenol-lyase (TPL) fra catechol, pyruvat og ammoniak, og aminosyre er direkte indarbejdet i proteiner af den genetiske indarbejdelse metode ved hjælp af en udviklet aminoacyl-tRNA og aminoacyl-tRNA syntetase par. Denne direkte iblanding system inkorporerer effektivt DOPA med lille inkorporering af andre naturlige aminosyrer og med bedre protein udbytte end den tidligere genetiske indarbejdelse system for DOPA. Protein konjugering med DOPA-indeholdende proteiner er effektiv og lokationsspecifikke og viser sin anvendelighed til forskellige applikationer. Denne protokol giver protein forskere med detaljerede procedurer for effektiv biosyntesen af mutant proteiner der indeholder DOPA på ønskede sites og deres konjugering for industrielle og farmaceutiske anvendelser.
Introduction
DOPA er en aminosyre, der er involverede i biosyntesen af katekolaminer i dyr og planter. Denne aminosyre er syntetiseret fra Tyr af tyrosin hydroxylase og molekylær ilt (O2)1. Fordi DOPA er en forløber for dopamin og kan præge blod – hjerne barrieren, har det været brugt i behandlingen af Parkinsons sygdom2. DOPA er også fundet i mussel vedhæftning proteiner (kort), som er ansvarlige for de klæbende egenskaber af muslinger i våde forhold3,4,5,6,7. Tyr er oprindeligt kodet på positioner hvor DOPA er fundet i MAPs og omdannes derefter til DOPA af tyrosinases8,9. På grund af sin interessante biokemiske egenskaber, er DOPA blevet brugt i en lang række applikationer. Dihydroxyl gruppen af DOPA er kemisk udsat for oxidation, og aminosyre er let omdannes til L-dopaquinone, en forløber for melaninas. På grund af sin høje electrofilia, har L-dopaquinone og derivater deraf været brugt til crosslinking og konjugering med dithioler og aminer10,11,12,13. 1,2-quinones kan også fungere som en Dien for cycloaddition reaktioner og har været brugt til bioconjugation af stamme-forfremmet oxidation-kontrollerede cyclooctyne-1,2-Quinon (SPOCQ) cycloaddition14. Derudover gruppen dihydroxyl kan Chelat metalioner som Fe3 + og Cu2 +, og proteiner der indeholder DOPA har været udnyttet til medicinafgivelse og metal-ion sensing15,16.
DOPA har været genetisk indarbejdet i proteiner ved hjælp af en ortogonal aminoacyl-tRNA (aa-tRNA) og aminoacyl-tRNA syntetase (aaRS) par17 og anvendes til protein konjugering og crosslinking10,11, 12,13. I denne betænkning, er eksperimentelle resultater og protokoller for den genetiske indarbejdelse af DOPA biosynthesized fra billige råvarer og dets applikationer til bioconjugation beskrevet. DOPA er biosynthesized ved hjælp af en TPL og start fra catechol, pyruvat og ammoniak i Escherichia coli. Biosynthesized DOPA er direkte indarbejdet i proteiner at give udtryk for en udviklet aa-tRNA og aaRS par for DOPA. Derudover er biosynthesized protein som indeholder DOPA site-specifically konjugeret med et fluorescerende sonde og crosslinked at producere protein oligomerer. Denne protokol vil være nyttigt for protein forskere, at biosynthesize mutant proteiner der indeholder DOPA og konjugat proteiner med biokemiske sonder eller narkotika for industrielle og farmaceutiske anvendelser.
Protocol
Representative Results
Discussion
I denne protokol, er biosyntese og direkte iblanding af DOPA beskrevet. Den bakterielle celle i denne metode kan syntetisere en yderligere aminosyre og bruge det som en unaturlig byggesten for proteinsyntese. Den genetiske indarbejdelse af unaturlige aminosyrer har været en nøgleteknologi til udvikling af unaturlige organisme med en udvidet genetiske kode. Dog, denne metode har været teknisk ufuldstændige og ændres for at forbedre indarbejdelse effektivitet og minimere undertrykkelse af netbårne til endogene maskin…
Declarações
The authors have nothing to disclose.
Acknowledgements
Denne forskning blev støttet af den globale grænse Research Program (NRF-2015M3A6A8065833), og den grundlæggende videnskab forskningsprogram (2018R1A6A1A03024940) gennem National Research Foundation af Korea (NRF) finansieret af Korea regering.
Materials
| 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 |
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