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Biochimica

OaAEP1-Medieret enzymatisk syntese og immobilisering af polymeriseret protein til enkeltmolekylet spektroskopi

Published: February 05, 2020
doi:
10.3791/60774
, , , , , ,
1State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering,Nanjing University

Summary

Her præsenterer vi en protokol til konjugere proteinmonomer af enzymer, der danner proteinpolymer med en kontrolleret sekvens og immobiliserer den på overfladen til enkeltmolekylets gennemspektroskopiundersøgelser.

Abstract

Kemiske og bio-bøjning teknikker er blevet udviklet hurtigt i de seneste år og tillader opførelse af protein polymerer. Men en kontrolleret protein polymerisering proces er altid en udfordring. Her har vi udviklet en enzymatisk metode til konstruktion af polymeriseret protein trin for trin i en rationelt kontrolleret sekvens. I denne metode er C-terminus af et proteinmonomer NGL for proteinbøjning ved hjælp af OaAEP1(Oldenlandia affinis asparaginyl endopeptidases)1), mens N-terminus var en cleavable TEV (tobak etch virus) spaltning site plus en L (ENLYFQ / GL) for midlertidig N-terminal beskyttelse. Derfor var OaAEP1 i stand til kun at tilføje ét proteinmonomer ad gangen, og derefter kløvede TEV N-endeinusmellem Q og G for at afsløre NH 2-Gly-Leu. Derefter er enheden klar til næste OaAEP1 ligation. Det manipulerede polyprotein undersøges ved at udfolde individuelle proteindomæne ved hjælp af atomkraftmikroskopibaseret single-molecule force spektroskopi (AFM-SMFS). Derfor, denne undersøgelse giver en nyttig strategi for polyprotein engineering og immobilisering.

Introduction

Sammenlignet med syntetiske polymerer har naturlige multidomæneproteiner en ensartet struktur med et velkontrolleret antal og typen af underdomæner1. Denne funktion fører normalt til forbedret biologisk funktion og stabilitet2,3. Mange tilgange, såsom cystein-baserede disulfid binding kobling og rekombinant DNA-teknologi, er blevet udviklet til at opbygge en sådan polymeriseret protein med flere domæner4,5,6,7. Men den tidligere metode resulterer altid i en tilfældig og ukontrolleret sekvens, og sidstnævnte fører til andre problemer, herunder vanskeligheden for overekspression af giftige og store proteiner og rensning af komplekst protein med cofaktor og andre sarte enzymer.

For at imødekomme denne udfordring udvikler vi en enzymatisk metode, der sammenlægger proteinmonomer sammen for polymer/polyprotein på en trinvis måde ved hjælp af en proteinligase OaAEP1 kombineret med en protease TEV8,9. OaAEP1 er en streng og effektiv endopeptidase. To proteiner kan forbindes kovalent som Asn-Gly-Leu sekvens (NGL) gennem to termini af OaAEP1 på mindre end 30min. Men brugen af OaAEP1 kun at forbinde protein monomer fører til et protein polymer med en ukontrolleret sekvens som cystein-baserede kobling metode. Derfor designer vi N-endepunktet af proteinenheden med et aftageligt TEV protease-sted plus en leucinrester som ENLYFQ/G-L-POI. Før TEV-kavalergangen ville N-terminalen ikke deltage i OaAEP1 ligation. Og så eksponeres GL-resterne ved N-endeinus, som er forenelige med yderligere OaAEP1 ligation, efter TEV-kavalergangen. Således har vi opnået en sekventiel enzymatisk biosyntese metode til polyprotein med en relativt velkontrolleret sekvens.

Her kan vores trinvise enzymatiske syntesemetode anvendes i polyproteinprøvepræparat, herunder sekvenskontrolleret og ukontrolleret, og proteinimmobilisering til enkeltmolekyleundersøgelser, især til det komplekse system som f.eks. metalloprotein.

Desuden giver AFM-baserede SMFS-eksperimenter os mulighed for at bekræfte proteinpolymerkonstruktionen og stabiliteten på enkeltmolekyleniveau. Single-molekyle kraft spektroskopi, herunder AFM, optisk pincet og magnetisk pincet, er et generelt redskab i nanoteknologi til at manipulere biomolekyle mekanisk og måle deres stabilitet11,12,13,14,15,16,17,18,19,20. Single-molecule AFM har været meget udbredt i studiet af protein (un)foldning21,22,23,24,25, styrken måling af receptor-ligand interaktion26,27,28,29,30,31,32,33,34 ,34, 35, uorganisk kemisk binding20,36,37,38,39,40,41,42,43 og metal-ligand binding i metalloprotein44,45,46,47,48,49,50 . Her anvendes single-molecule AFM til at verificere den syntetiserede polyproteinsekvens baseret på det tilsvarende proteinudfoldersignal.

Protocol

1. Proteinproduktion Genkloning Køb gener kodning for protein af interesse (POI): Ubiquitin, Rubredoxin (RD)51, cellulose-bindende modul (CBM), dockerin-X domæne (XDoc) og samhørighed fra Ruminococcus flavefacience, tobak etch virus (TEV) protease, elastin-lignende polypeptider (ELP’ er). Udfør polymerase kædereaktion og bruge tre-begrænsning fordøjelseenzym system BamHI-BglII-KpnI for rekombinere genet fra…

Representative Results

De NGL-restkoncentrationer, der indføres mellem tilstødende proteiner ved OaAEP1 ligation, vil ikke påvirke proteinmonomerstabiliteten i polymeren som udfoldelseskraft (<Fu>), og konturlængdeforøgelsen (<ΔLc>) kan sammenlignes med den foregående undersøgelse (figur 1). Rensningsresultatet af rubredoxinproteinet er vist i figur 2. For at bevise, at proteinet efter TEV spaltning er kompatibelt med følgende OaAEP1 ligation til at kons…

Discussion

Vi har beskrevet en protokol for enzymatisk biosyntese og immobilisering af polyprotein og verificeret polyprotein design af AFM-baserede SMFS. Denne metode giver en ny tilgang til opbygning af protein-polymer i en designet sekvens, som supplerer tidligere metoder til polyprotein engineering og immobilisering4,6,52,53,54,55,<…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

Dette arbejde blev støttet af National Natural Science Foundation of China (Grant No. 21771103, 21977047), Natural Science Foundation of Jiangsu-provinsen (Grant No. BK20160639) og Shuangchuang Program i Jiangsu-provinsen.

Materials

iron (III) chloride hexahydrate Energy chemical 99%
Zinc chloride Alfa Aesar 100.00%
calcium chloride hydrate Alfa Aesar 99.9965% crystalline aggregate
L-Ascorbic Acid Sigma Life Science Bio Xtra, ≥99.0%, crystalline
(3-Aminopropyl) triethoxysilane Sigma-Aldrich ≥99%
sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate Thermo Scientific 90%
Glycerol Macklin 99%
5,5'-dithiobis(2-nitrobenzoic acid) Alfa Aesar
Genes Genscript
Equipment
Nanowizard 4 AFM JPK Germany
MLCT cantilever Bruker Corp
Mono Q 5/50 GL GE Healthcare
AKTA FPLC system GE Healthcare
Glass coverslip Sail Brand
Nanodrop 2000 Thermo Scientific
Avanti JXN-30 Centrifuge Beckman Coulter
Gel Image System Tanon
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Tags

OaAEP1Enzymatic SynthesisPolymerized ProteinSingle-molecule Force SpectroscopyProtein OligomerPolymerization DegreeProtein-based MaterialCysteinePotassium ChromateSulfuric AcidAPTESSulfo-SMCCGL-ELP-50 Cys-proteinCantilever Surface
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Citazione di questo articolo
Deng, Y., Zheng, B., Liu, Y., Shi, S., Nie, J., Wu, T., Zheng, P. OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy. J. Vis. Exp. (156), e60774, doi:10.3791/60774 (2020).
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