Cell-free protein expression has become a popular tool in many biology fields ranging from synthetic biology to industrial biomanufacturing.
Compared to in vivo approaches, cell-free protein expression allows rapid and inexpensive protein production with the highest versatility and flexibility due to its open-reaction environment as it completely lacks cells walls and membranes allowing easy access to all cellular processes. Thus efficient substrate handling, precise online reaction monitoring and direct process optimization processes is conveniently promoted.
Rely on the experts at Arbor Biosciences for a broad knowledge of DNA synthesis and cloning, combined with a versatile and efficient in vitro protein expression platform under a single roof to provide unique and comprehensive solutions for custom projects or specialized application.
Tired of spending days on transformation as well as elaborate cell cultivation and processing to get to your protein of interest? Are you looking for a cost-effective protein production platform in this fast-paced world where time is money? Cell-free protein expression is the answer to accelerate your research enabling you to easily manipulate reaction parameters and gain full control over the supply of cost-intensive substrates in a much faster and effective rate compared to conventional cell-based methods.
myTXTL® relies on the endogenous transcription and translation machinery of E. coli, making use of the core RNA polymerase and primary sigma factor 70 (σ70) found in its cytoplasm. The myTXTL Sigma 70 Master Mix contains all biological components required for transcription, translation, protein folding and energy regeneration, such as ribosomes, tRNAs, chaperones, metabolic enzymes, elongation and translation initiation factors, in a single tube to which you simply add the plasmid DNA template for expression of your protein of interest. All genes should be under the control of a σ70-specific promoter such as the promoter found in our P70a vector. Gene expression with a T7 promoter requires the co-expression of the T7 RNA polymerase under the control of a σ70-specific promoter, for example P70a-T7rnap.
Difficult to Express Proteins
myTXTL has been successfully used to synthesize proteins over a wide range of molecular weight and consisting of single or multiple domains that were proven to be functionally active. Examples include a palette of fluorescent reporter proteins like eGFP, approximately 25 kDa single domain, that undergo a multi-step maturation process to obtain their active state; firefly luciferase as a two-domain enzyme of about 60 kDa that catalyzes the oxygenation of luciferin, a light emitting compound; and T7 and T3 RNA polymerase being multi-domain proteins around 100 kDa responsible for gene transcription. Moreover, cell-free systems are a perfect choice if undesired cell lysis during cell cultivation resulting from a leaky promoter or cytotoxic gene product diminishes protein yield. Decoupling of cell growth from protein over-expression considerably increases the range of proteins that can be recombinantly expressed, such as proteins that interfere with cell wall synthesis or require a specific heavy metal for their functionality, like enzymes. Hereby, the myTXTL Sigma 70 Master Mix facilitates expression of eukaryotic proteins as wells as prevents undesired translation stop as it contains tRNAs for seven codons (AGA, AGG, AUA, CUA, GGA, CCC, and CGG) rarely used in E. coli..
Membrane proteins are challenging targets for any translation system. Due to the enhanced control over protein synthesis in vitro, considerably higher yields of soluble, active membrane proteins can be generated compared to cell-based approaches. Newly synthesized membrane proteins can be stabilized by adding membrane mimics, for example surfactants, liposomes or nanodiscs, to the cell-free reaction either post-translationally for protein solubilization, or co-translationally, to prevent aggregation. Please refer to publications using our myTXTL® cell-free system for membrane protein synthesis.
Expression of Proteins with Disulfide Bonds
Although the currently available Sigma 70 Master Mix is not optimized to produce disulfide bond containing proteins, studies have shown similar strategies utilized for in vivo expression systems can promote correct formation of disulfide bonds in vitro. These include supplementing cell-free systems with mixtures of reduced (GSH) and oxidized glutathione (GSSG), disulfide bond isomerase C (DsbC), protein disulfide isomerase (PDI) with potential chaperones such as DnaK, DnaJ, GroEL, GroES. In addition, pretreatment with iodoacetamide (IAM) to inactivate endogenous reductases which are present in the cell extract has proven to be beneficial.
Marshall, R. et al. (2018). Rapid and scalable characterization of CRISPR technologies using an E. coli cell-free transcription-translation system. Molecular Cell.
Maxwell, C.S. et al. (2018) A detailed cell-free transcription-translation-based assay to decipher CRISPR protospacer-adjacent motifs. Methods.
Rustad, M. et al. (2018). Cell-free TXTL synthesis of infectious bacteriophage T4 in a single test tube reaction. Synthetic Biology.
Majumder, S. et al. (2017). Cell-sized mechanosensitive and biosensing compartment programmed with DNA. Chemical Communications (Cambridge, England).
Garamella, J. et al. (2016). The all E. coli TX-TL toolbox 2.0: a platform for cell-free synthetic biology. ACS Synth Biol.
Coutable, A. et al. (2014) Preparation of tethered-lipid bilayers on gold surfaces for the incorporation of integral membrane proteins synthesized by cell-free expression. Langmuir.