Tobacco BY-2 cell-free lysate: an alternative and highly-productive plant-based in vitro translation systemReportar como inadecuado




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BMC Biotechnology

, 14:37

Protein and enzyme technology

Abstract

BackgroundCell-free protein synthesis is a rapid and efficient method for the production of recombinant proteins. Usage of prokaryotic cell-free extracts often leads to non-functional proteins. Eukaryotic counterparts such as wheat germ extract WGE and rabbit reticulocyte lysate RLL may improve solubility and promote the correct folding of eukaryotic multi-domain proteins that are difficult to express in bacteria. However, the preparation of WGEs is complex and time-consuming, whereas RLLs suffer from low yields. Here we report the development of a novel cell-free system based on tobacco Bright Yellow 2 BY-2 cells harvested in the exponential growth phase.

ResultsThe highly-productive BY-2 lysate BYL can be prepared quickly within 4–5 h, compared to 4–5 d for WGE. The efficiency of the BYL was tested using three model proteins: enhanced yellow fluorescent protein eYFP and two versions of luciferase. The added mRNA was optimized by testing different 5’ and 3’ untranslated regions UTRs. The protein yield in batch and dialysis reactions using BYL was much higher than that of a commercial Promega WGE preparation, achieving a maximum yield of 80 μg-mL of eYFP and 100 μg-mL of luciferase, compared to only 45 μg-mL of eYFP and 35 μg-mL of luciferase in WGEs. In dialysis reactions, the BYL yielded about 400 μg-mL eYFP, representing up to 50% more of the target protein than the Promega WGE, and equivalent to the amount using 5Prime WGE system.

ConclusionsDue to the high yield and the short preparation time the BYL represents a remarkable improvement over current eukaryotic cell-free systems.

KeywordsCell-free protein synthesis In vitro translation Nicotiana tabacum cv. BY-2 Wheat germ extract Protein expression AbbreviationsBYDFBarley yellow dwarf virus

BYLBY-2 lysate

CECFContinuous exchange cell-free

CFPSCell-free protein synthesis

EMCVEncephalomyocarditis virus

eYFPEnhanced yellow fluorescent protein

FFLucFirefly luciferase

ICEInsect cell extract

IMACImmobilized metal-affinity chromatography

RLLRabbit reticulocyte lysate

RRLucRenilla reniformis luciferase

SECSize exclusion chromatography

TMVTobacco mosaic virus

UTRUntranslated region

WGEWheat germ extract.

Electronic supplementary materialThe online version of this article doi:10.1186-1472-6750-14-37 contains supplementary material, which is available to authorized users.

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Autor: Matthias Buntru - Simon Vogel - Holger Spiegel - Stefan Schillberg

Fuente: https://link.springer.com/



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