Basic Principles of Translation Biology
The portfolio of today’s existing systems, though extending over a broad and diverse spectrum, all fall under one of the two types of cell-free expression systems: Translation Systems and Coupled Transcription and Translation (TnT®) Systems. Despite the differences between the systems, the basic principles of the cell-free reaction remain the same.
Cell-free expression begins with crude extracts generated from cultured cells that are typically engaged in a high rate of protein synthesis, such as immature red blood cells (reticulocytes). These crude extracts are depleted of their endogenous DNA and mRNA, and the cell lysate is subsequently supplemented with macromolecular components required to perform translation, including ribosomes, tRNAs, aminoacyl-tRNA synthetases and initiation, elongation and termination factors. The process of translation is then initiated by adding a suitable template (DNA or mRNA) and carried out at an appropriate temperature. In Translation Systems, reactions are initiated with purified mRNA, while systems that are initiated with linear or plasmid DNA templates are referred to as Coupled Transcription and Translation (TnT®) Systems.
To ensure efficient translation, each extract requires additional supplementation with amino acids, energy sources (ATP, GTP), energy regenerating systems and salts (e.g., Mg2+, K+). Creatine phosphate and creatine phosphokinase typically serve as energy regenerating systems in eukaryotic systems, whereas prokaryotic systems are often supplemented with phosphoenolpyruvate and pyruvate kinase. Additionally, coupled transcription and translation systems are also typically supplied with phage-derived RNA polymerase (T3, T7 or SP6), which transcribes mRNA from an exogenous DNA template and allows for expression of genes cloned downstream of a T3, T7 or SP6 promoter.
Cell Extract and System Selection
When it comes down to selecting the right cell-free protein expression system for you, there are several factors that need to be taken into consideration, including the type of template you will be using, your desired protein yield and the intended downstream applications.
The most popular commercially-available in vitro translation systems to date consist of E. coli, wheat germ, rabbit reticulocytes or insect cell extracts. As each of these cells behave and function in different ways, the same is also true of their derived extracts, and each have their own advantages and disadvantages, which are briefly highlighted below (1; 3).
The prokaryotic E. coli system is currently the most popular protein expression system for several reasons. E. coli extraction preparation is simple and inexpensive, as E. coli is easily fermented in large quantities utilizing low-cost media and is ruptured easily using high-pressure homogenizers. E. coli-based systems also generally achieve the highest protein yields, and the total reaction cost of E. coli system is the collective lowest. E. coli is capable of activating metabolic reactions in the extract which in turn fuels high-level protein synthesis, which eliminates the need for more expensive energy substrates like phosphoenolpyruvate.
The Wheat Germ Extract (WGE), Rabbit Reticulocyte Lysate (RRL) and Insect Cell Extract (ICE) systems are currently the most widely used eukaryotic systems. These systems are advantageous in production of more complex proteins, and can also achieve post-translational modifications that are not found in E. coli . However, these eukaryotic systems do generally involve more laborious extract preparation procedures, which can increase cost. The eukaryotic systems also tend to result in lower protein yields in batch reactions when compared to E. coli systems.
Cell-free extracts of wheat germ and rabbit reticulocyte lysate support the in vitro translation of a wide variety of viral, prokaryotic and eukaryotic mRNAs. These RNA-driven systems are widely used to identify mRNA species and characterize their products. Starting with the DNA of interest, in vitro transcripts (5–80µg/ml) for translation can be generated with the RiboMAX™ Large Scale RNA Production Systems (Cat.# P1280, P1300) and the T7 RiboMAX™ Express Large Scale RNA Production System (Cat.# P1320).
E. coli S30 Extract (ECE):
Advantages: The extraction preparation is both simple and cost-effective. ECE systems have the capability of folding complex proteins, consistently have a high rate of protein synthesis and a resulting high protein synthesis yield. Additionally, the energy sources are low-cost, the current methods are well-understood and there are many well-established tools available to perform genetic modifications as well.
Disadvantages: The number of optional post-translational modifications is limited, and there are no endogenous membrane structures for synthesis of integral membrane proteins.
Wheat Germ Extract (WGE):
Advantages: A wide-spectrum expression of eukaryotic proteins have been achieved repeatedly utilizing WGE. This is also a highly productive system, which translates into a high yield of complex proteins. Sophisticated high-throughput method for proteomics.
Disadvantages: The lysate preparation can be expensive and labor-intensive. Limited post-translational modifications are possible, there are no endogenous membrane structures for synthesis of integral membrane proteins and WGE offers a low protein yield in comparison to prokaryotic systems.
Rabbit Reticulocyte Lysates (RRL):
Advantages: Cells are easy to break and the extraction preparation process is quick. The RRL system is a tried-and-true, well-established system. This system is well-suited for mammalian system eukaryotic-specific modifications, with moderate/low yields for protein.
Disadvantages: Low protein yield. Post-translational modifications only possible by supplementing with exogenous microsomal membranes.
Insect Cell Extract (ICE):
Advantages: Cells are easy to break and the extraction preparation process is quick. Many eukaryotic-specific post-translational modifications are possible using this extract, including glycosylation, disulfide-bridge formation, lipidation, and signal peptide cleavage phosphorylation. Endogenous microsomes are also available, and direct synthesis and integration of membrane proteins has been performed successfully using this extract.
Disadvantages: Insect cell extracts tend to have high cultivation costs.
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Advantages of Cell-Free Protein Expression Systems
Cell-free protein expression systems offer several distinct advantages in comparison to cell-based protein expression, including increased overall yields of full-length proteins that are both functional and soluble, as well as considerable time savings. Typical in vivo methods can take, at best, days and at worst, weeks, to complete (4). In stark contrast, in vitro translation reactions, including the time invested in preparation of extracts, can be performed in only a few hours, providing the fastest way to correlate phenotype (the function of expressed protein) to genotype.
Utilizing a cell-free approach, protein synthesis is also versatile in that it can be performed using a variety of inputs. Cell-free translational systems utilize mRNA as a template, while either plasmid DNA or linear PCR fragments can serve as a DNA template in coupled transcription/translation systems.