A versatile coupled cell‐free transcription–translation system based on tobacco BY‐2 cell lysates

Abstract

Cell-free protein synthesis is a powerful method for the high-throughput production of recombinant proteins, especially proteins that are difficult to express in living cells. Here we describe a coupled cell-free transcription-translation system based on tobacco BY-2 cell lysates (BYLs). Using a combination of fractional factorial designs and response surface models, we developed a cap-independent system that produces more than 250 μg/mL of functional enhanced yellow fluorescent protein (eYFP) and about 270 μg/mL of firefly luciferase using plasmid templates, and up to 180 μg/mL eYFP using linear templates (PCR products) in 18 h batch reactions. The BYL contains actively-translocating microsomal vesicles derived from the endoplasmic reticulum, promoting the formation of disulfide bonds, glycosylation and the cotranslational integration of membrane proteins. This was demonstrated by expressing a functional full-size antibody (∼ 150 μg/mL), the model enzyme glucose oxidase (GOx) (∼ 7.3 U/mL), and a transmembrane growth factor (∼ 25 μg/mL). Subsequent in vitro treatment of GOx with peptide-N-glycosidase F confirmed the presence of N-glycans. Our results show that the BYL can be used as a high-throughput expression and screening platform that is particularly suitable for complex and cytotoxic proteins.

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

“…In principle, any organism can be used to provide a source of crude lysate. However, the most commonly used systems are the prokaryotic Escherichia coli system (bacterium) (Jewett and Swartz, 2004a;Kwon and Jewett, 2015), as well as the eukaryotic systems based on crude cell lysates from Saccharomyces cerevisiae (fungus) (Gan and Jewett, 2014;Hodgman and Jewett, 2013), wheat germ (plant) (Madin et al, 2000;Takai et al, 2010), tobacco (plant) (Buntru et al, 2015;Komoda et al, 2004), Spodoptera frugiperda (insect) (Stech et al, 2014;Tarui et al, 2001), rabbit reticulocytes (mammalian) (Anastasina et al, 2014;Pelham and Jackson, 1976) and Chinese hamster ovary (CHO, mammalian) (Br€ odel et al, 2014). These CFPS systems are developed for different biological and application-based goals, albeit each one has its own advantages and disadvantages (Zemella et al, 2015).…”

Versatile Cell-Free Protein Synthesis Systems Based on Chinese Hamster Ovary Cells

“…Special eukaryotic cell-free systems contain endogenous microsomes derived from the endoplasmic reticulum due to a mild cell disruption procedure during lysate preparation. Microsomes enable an integration of membrane embedded and associated proteins into a nature-like milieu, a cotranslational translocation of proteins as reported in cellular protein expression processes and they harbor a fully ER based set of enzymes for folding and posttranslational modifications including relevant glycosyltransferases for attachment of glycan moieties, chaperones and essential molecules for the introduction of disulfide bridges [11][12][13][14]. In order to combine a mammalian host cell often used for the production of versatile, mammalian proteins and a cell-free platform, we have recently developed novel systems based on the prominent industrial protein production host CHO cells [15,16].…”

ChemInform Abstract: Cell‐Free Protein Synthesis: Pros and Cons of Prokaryotic and Eukaryotic Systems

“…Af irst look at post-translational modificationsi ndicatedt hat glycosylations and disulfide bond formation are possible, thereby resulting in the synthesis of af unctionally active full-size antibody. [90] Neverthelessf urther optimization steps, scale-up, and functional analysiso fm ore complexp roteins are essential to obtainarobust protein productionsystem.…”