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

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.

Establishing a high yielding streptomyces‐based cell‐free protein synthesis system

“…We started with magnesium concentration, which is known to be a fundamentally important physicochemical salt used in cell-free systems that influences the functional activity of the translation apparatus (Jewett et al, 2008;Klein et al, 2004;Liiv and O'Connor, 2006;Yamamoto et al, 2010). Previous reports have shown that the optimal Mg 2þ concentrations in different CFPS systems are also different, for example, E. coli (12 mM) (Jewett and Swartz, 2004a), yeast (7 mM) (Hodgman and Jewett, 2013), Bacillus subtilis (15 mM) (Zaghloul and Doi, 1987), PURE system (9 mM) (Shimizu et al, 2001), wheat germ extract (2.5 mM) (Marcu and Dudock, 1974) and tobacco BY-2 extract (1.44 mM) (Buntru et al, 2014). Although the early study reported that 12 mM of Mg 2þ was optimal for the Streptomyces cell-free system (Thompson et al, 1984), we decided to re-examine the magnesium ion concentration.…”

Non-conventional expression systems for the production of vaccine proteins and immunotherapeutic molecules

“…152 In practice, however, only a few cell-free systems have been developed for in vitro protein synthesis. In general, these systems are derived from cells engaged in a high rate of protein synthesis, and commercially available systems exist for various applications including ribosome display and unnatural amino acid incorporation.…”

Comparison of cell‐based versus cell‐free mammalian systems for the production of a recombinant human bone morphogenic growth factor

“…Established eukaryotic systems include yeast [18], wheat germ [19,20], insect [21,22], tobacco [23] and mammalian [24,25] cell lysates. The wheat germ system is highly popular due to its high yields, but limited in regard to the correct post-translational modification of human proteins.…”