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Biochemistry

Biosynthesized l-dihydroxyphenylalanine (多巴) 的遗传合并及其在蛋白质共轭中的应用

Published: August 24, 2018
doi:
10.3791/58383
,
1Department of Chemistry,Sogang University

Summary

在此, 我们提出了一种从简单的起始材料中 dihydroxyphenylalanine biosynthesized 的遗传方法及其在蛋白质共轭中的应用的协议。

Abstract

l-dihydroxyphenylalanine (多巴) 是在动物和植物中儿茶酚胺的生物合成中发现的一种氨基酸。由于氨基酸具有特殊的生化特性, 在生物化学应用中具有多种用途。本报告描述了 biosynthesized 巴的遗传合并协议及其在蛋白质共轭中的应用。多巴是由邻苯二酚、丙酮酸和氨的酪氨酸酚裂解酶 (TPL) biosynthesized 的, 而氨基酸则采用进化后的氨酰-tRNA 和氨酰-tRNA 合成产物对的遗传法直接纳入蛋白质中。这种直接的并网系统有效地吸收了多巴与其他天然氨基酸的很少结合, 并具有更好的蛋白质产量比以前的多巴基因合并系统。多巴多巴蛋白结合具有高效性和特异性, 并显示其对各种应用的实用性。该议定书为蛋白质科学家提供了在所需地点对含有巴胺的突变蛋白进行有效生物合成的详细程序, 以及它们用于工业和制药应用的共轭。

Introduction

巴胺是一种氨基酸, 涉及动物和植物中儿茶酚的生物合成。该氨基酸由酪氨酸羟化酶和分子氧 (O2)1合成 Tyr。由于多巴是一大胺的前驱体, 可以渗透血脑屏障, 它已用于治疗帕金森病2。巴比在贻贝黏附蛋白 (地图) 也被发现, 负责贻贝的胶粘剂物产在湿的情况3,4,5,6,7。Tyr 最初是在地图上发现了几巴的位置编码的, 然后被 tyrosinases8、9转换成了巴比。多巴由于其具有有趣的生化特性, 在各种应用中得到了应用。多巴二羟基组化学上易氧化, 氨基酸容易转化为 l-dopaquinone, 黑色素的前驱体。由于其高 electrophilicity, l-dopaquinone 及其衍生物已被用于交联和共轭与硫醇和胺10,11,12,13。12-醌还可以作为 cycloaddition 反应的二烯, 并已用于 bioconjugation 由应变促进氧化控制 cyclooctyne-1,2-quinone (SPOCQ) cycloaddition14。此外, 二羟基集团可以螯合金属离子, 如 Fe3 +和铜2 +, 和含有多巴的蛋白质已用于药物输送和金属离子传感15,16。

多巴通过使用正交氨酰-tRNA (aa-tRNA) 和氨酰-tRNA 合成酶 (aaRS) 对17 , 并用于蛋白质共轭和交联10,11, 被基因纳入蛋白质,12,13。在本报告中, 介绍了从廉价的起始材料中引入 biosynthesized 的实验结果和协议, 以及它在 bioconjugation 中的应用。biosynthesized 使用 TPL, 从邻苯二酚, 丙酮酸和氨的大肠杆菌开始。biosynthesized 多巴是通过表达一个进化的 aa-tRNA 和 aaRS 对巴比直接纳入蛋白质。此外, 含有多巴的 biosynthesized 蛋白与荧光探针结合, 并交联以产生蛋白质寡聚物。这项协议将是有用的蛋白质科学家, biosynthesize 突变蛋白含有一巴和共轭蛋白质与生化探针或药物的工业和制药应用。

Protocol

1. 质粒结构 构建一种表达质粒 (pBAD-双 TPL-gfp), 在本构启动子和绿色荧光蛋白 (gfp) 基因的控制下表达柠檬酸杆菌 freundii的 TPL 基因, 并在其控制下对其6标记araBAD 启动子。对于 pBAD-dual-TPL-GFP-E90TAG, 使用站点定向的突变协议, 用琥珀密码子 (标记) 替换多巴的站点 (E90) 的密码子。我们在上一份报告18中描述了建造这种质粒的细节。 构建 tRNA/aaRS 表达质粒 (pE…

Representative Results

图 1显示了从 TPL 直接并入 biosynthesized 的表达系统。进化的 aa-tRNA 和 aaRS 对的基因被放置在质粒中, 而 gfp 基因 (GFP-E90TAG) 包含琥珀密码子在位置90位于另一个质粒中, 以评估多巴的加入 gfp 荧光。TPL 基因被放置在含有 GFP 基因和组成性表达的同一个表达质粒中, 以最大程度地提高巴比生物合成的产量。利用该表达系统筛选了巴比生物合成的最佳条件。本…

Discussion

在本议定书中, 介绍了巴胺的生物合成和直接加入。这种方法中使用的细菌细胞可以合成额外的氨基酸, 并将其用作蛋白质合成的非自然构造块。非自然氨基酸的遗传融合一直是生物进化的关键技术, 具有扩展的遗传密码。然而, 这种方法在技术上是不完整的, 正在修改, 以提高整合效率, 并尽量减少扰动的内源翻译系统。最近, 该方法取得了重大进展, 通过重新分配密码子 24,</su…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项研究得到了全球前沿研究项目 (NRF-2015M3A6A8065833) 的支持, 并通过韩国政府资助的韩国国家研究基金会 (2018R1A6A1A03024940) (NRF) 进行了基础科学研究项目。

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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Tags

Genetic IncorporationL-dihydroxyphenylalanine (DOPA)Protein ConjugationBiosynthetic SystemE. ColiExpression PlasmidElectroporationLB MediumPA-5052 MediumCell LysisProtein Purification
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Kim, S., Lee, H. S. Genetic Incorporation of Biosynthesized L-dihydroxyphenylalanine (DOPA) and Its Application to Protein Conjugation. J. Vis. Exp. (138), e58383, doi:10.3791/58383 (2018).
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