Cell-free system

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A cell-free system is an in vitro tool widely used to study biological reactions that happen within cells apart from a full cell system, thus reducing the complex interactions typically found when working in a whole cell. [1] Subcellular fractions can be isolated by ultracentrifugation to provide molecular machinery that can be used in reactions in the absence of many of the other cellular components. [2] Eukaryotic and prokaryotic cell internals have been used for creation of these simplified environments. [3] [4] These systems have enabled cell-free synthetic biology to emerge, providing control over what reaction is being examined, as well as its yield, and lessening the considerations otherwise invoked when working with more sensitive live cells. [5]

Contents

Types

Cell-free systems may be divided into two primary classifications: cell extract-based, which remove components from within a whole cell for external use, and purified enzyme-based, which use purified components of the molecules known to be involved in a given process. [6] [7] The cell extract-based type are susceptible to problems like quick degradation of components outside their host, as shown in a study by Kitaoka et al. where a cell-free translation system based on Escherichia coli (E. coli), of the cell extract-based type, had the mRNA template degrade very quickly and led to the halt of protein synthesis. [8]

Preparation

The methods of preparation vary between situations of both types of cell-free systems.

Cell extract–based

Nobel prize winner Eduard Buchner was arguably the first to present a cell-free system using yeast extracts, but since then alternative sources have been found. [9] [10] E. coli, wheat germ, and rabbit reticulocytes have all proven useful to create cell-free systems by extraction of their interior components. [3] [11] E. coli 30S extracts have been acquired, for example, by grinding the bacteria with alumina, followed by further cleaning. [12] Similarly, wheat germ has been ground with acid-washed sand or powdered glass to open the cell membranes up. [13] [14] Rabbit reticulocytes have been lysed in a solution of MgCl2 and had the extract filtered away from the membranes by centrifugation. [15]

Uses

Cell-free synthetic pathway biotransformation biosystems are proposed as a new low-cost biomanufacturing platform compared to microbial fermentation used for thousands of years. [3] [16] Cell-free biosystems have several advantages suitable in industrial applications: [6]

C6H10O5 (l) + 7 H2O (l) → 12 H2 (g) + 6 CO2 (g),
nearly 12 H2 has been produced per glucose unit of polysaccharides and water, three times of the theoretical yield of the best anaerobic hydrogen-producing microorganisms. [17]

Protein synthesis

In vitro biosystems can be easily controlled and accessed without membranes. [16] Notably, in work leading to a Nobel prize the Nirenberg and Matthaei experiment used a cell-free system, of the cell extract-based type, to incorporate chosen amino acids tagged radioactively into synthesized proteins with 30S extracted from E. coli. [12] [21] More recent studies, such as the study done by Spirin et al. with prokaryotic and eukaryotic version of their cell-free translation system, have also synthesized proteins with increased production, incorporating techniques like continuous flow to add materials and remove products. [22] With such advances in yield, productivity applications have been expanded, such as the synthesis of fusion proteins to potentially serve as vaccines for B-cell lymphomas. [23] Additionally, cell-free protein synthesis is becoming a new alternative choice for fast protein synthesis. [6]

Metabolic manipulation

Engineering of metabolic processes have been achieved through cell-free systems. [24] [10] [3] Bujara et al., for example, were able to use glycolytic network extracts, consisting of enzymes from E. coli that produced dihydroxyacetone phosphate, to analyze in real-time the metabolite concentrations while altering enzyme levels, with the end result of optimal production of dihydroxyacetone phosphate. [25] Further, Calhoun and Swartz were able to use a glycolytic intermediate to fuel a cell-free system, enabling relatively inexpensive ATP generation compared to reagent usage in phosphoenolpyruvate reactions. [26]

Unnatural amino acid incorporation

Cell-free systems have also been used to incorporate unnatural amino acids. [26] [27] Shimizu et al. were able to change a stop codon to a sense codon by omitting the RF1 release factor, indicating ability to insert desired amino acids in unnatural situations. This is of use in systems where working inside a cell is problematic, such as the process of amino acid metabolism preventing specific labelling of amino acids that would be useful in multidimensional NMR spectroscopy. [28] Kigawa et al.were able to successfully label amino acids in a cell-free system where amino acid metabolism was no longer present, thus making such systems useful to NMR studies. [28]

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<span class="mw-page-title-main">Nediljko Budisa</span>

Nediljko "Ned" Budisa is a Croatian biochemist, professor and holder of the Tier 1 Canada Research Chair (CRC) for chemical synthetic biology at the University of Manitoba. As pioneer in the areas of genetic code engineering and chemical synthetic biology (Xenobiology), his research has a wide range of applications in biotechnology and engineering biology in general. Being highly interdisciplinary, it includes bioorganic and medical chemistry, structural biology, biophysics and molecular biotechnology as well as metabolic and biomaterial engineering. He is the author of the only textbook in his research field: “Engineering the genetic code: expanding the amino acid repertoire for the design of novel proteins”.

Dr. Herbert Weissbach NAS NAI AAM is an American biochemist/molecular biologist.

References

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