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A User's Guide to Cell-Free Protein Synthesis

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A User's Guide to Cell-Free Protein Synthesis Review A User’s Guide to Cell-Free Protein Synthesis Nicole E. Gregorio 1,2 , Max Z. Levine 1,3 and Javin P. Oza 1,2,* 1 Center for Applications in Biotechnology, California Polytechnic State University, San Luis Obispo, CA 93407, USA; [email protected] (N.E.G.); [email protected] (M.Z.L.) 2 Department of Chemistry and Biochemistry, California Polytechnic State University, San Luis Obispo, CA 93407, USA 3 Department of Biological Sciences, California Polytechnic State University, San Luis Obispo, CA 93407, USA * Correspondence: [email protected]; Tel.: +1-805-756-2265 Received: 15 February 2019; Accepted: 6 March 2019; Published: 12 March 2019 Abstract: Cell-free protein synthesis (CFPS) is a platform technology that provides new opportunities for protein expression, metabolic engineering, therapeutic development, education, and more. The advantages of CFPS over in vivo protein expression include its open system, the elimination of reliance on living cells, and the ability to focus all system energy on production of the protein of interest. Over the last 60 years, the CFPS platform has grown and diversified greatly, and it continues to evolve today. Both new applications and new types of extracts based on a variety of organisms are current areas of development. However, new users interested in CFPS may find it challenging to implement a cell-free platform in their laboratory due to the technical and functional considerations involved in choosing and executing a platform that best suits their needs. Here we hope to reduce this barrier to implementing CFPS by clarifying the similarities and differences amongst cell-free platforms, highlighting the various applications that have been accomplished in each of them, and detailing the main methodological and instrumental requirement for their preparation. Additionally, this review will help to contextualize the landscape of work that has been done using CFPS and showcase the diversity of applications that it enables. Keywords: cell-free protein synthesis (CFPS); in vitro transcription-translation (TX-TL); cell-free protein expression (CFPE); in vitro protein synthesis; cell-free synthetic biology; cell-free metabolic engineering (CFME) 1. Introduction Cell-free protein synthesis (CFPS) emerged about 60 years ago as a platform used by Nirenberg and Matthaei to decipher the genetic code and discover the link between mRNA and protein synthesis [1]. Since this discovery, the CFPS platform has grown to enable a variety of applications, from functional genomics to large-scale antibody production [2,3]. Currently, CFPS has been implemented using cell extracts from numerous different organisms, with their unique biochemistries enabling a broad set of applications. In an effort to assist the user in selecting the CFPS platform that is best suited to their experimental goals, this review provides an in-depth analysis of high adoption CFPS platforms in the scientific community, the applications that they enable, and methods to implement them. We also review applications enabled by low adoption platforms, including applications proposed in emerging platforms. We hope that this will simplify new users’ choice between platforms, thereby reducing the barrier to implementation and improving broader accessibility of the CFPS platform. The growing interest in CFPS is the result of the key advantages associated with the open nature of the platform. The CFPS reaction lacks a cellular membrane and a functional genome, and consequently is not constrained by the cell’s life objectives [4]. Therefore, the metabolic and cytotoxic burdens placed on the cell when attempting to produce large quantities of recombinant proteins in vivo are obviated Methods Protoc. 2019, 2, 24; doi:10.3390/mps2010024 www.mdpi.com/journal/mps Methods and Protoc. 2019, 2, 24 2 of 38 consequentlyMethods Protoc. 2019 is not, 2, 24 constrained by the cell’s life objectives [4]. Therefore, the metabolic and cytotoxic2 of 34 burdens placed on the cell when attempting to produce large quantities of recombinant proteins in vivo are obviated in CFPS [5]. The CFPS platform is amenable to direct manipulation of the environmentin CFPS [5]. Theof protein CFPS platformproduction is amenable because it to is direct an open manipulation system (Figure of the 1). environment In some cases of proteinhigher productionprotein titers because can be it isachieved an open using system CFPS (Figure because1). In someall energy cases higherin the proteinsystem titersis channeled can be achieved toward producingusing CFPS the because protein all of energy interest in (Figure the system 2) [6]. is Mo channeledreover, CFPS toward reactions producing are flexible the protein in their of interest setup, (Figureallowing2)[ users6]. Moreover, to utilize a CFPS variety reactions of reaction are flexible format ins, such their as setup, batch, allowing continuous users flow, to utilize and continuous a variety of exchange,reaction formats, in order such to asachieve batch, the continuous desired protein flow, and titer continuous (Figure 3). exchange, These advantages in order to make achieve CFPS the optimallydesired protein suited titer for (Figure applications3). These such advantages as the prod makeuction CFPS of optimally difficult-to-synthesize suited for applications proteins, such large as proteins,the production proteins of difficult-to-synthesizeencoded by high GC proteins,content ge largenes, proteins, membrane proteins proteins, encoded and byvirus-like high GC particles content (Figuregenes, membrane 4A & 5A). The proteins, scalable and nature virus-like of CFPS particles allows (Figures it to support4A and the5A). discovery The scalable phase nature through of CFPS high- throughputallows it to supportscreening the discoveryas well as phase the throughproduction high-throughput phase through screening large-scale as well biomanufacturing. as the production phaseAdditional through high large-scale impact applications biomanufacturing. include Additional education, high metabolic impact engineering, applications includeand genetic education, code expansion.metabolic engineering, and genetic code expansion. Figure 1. AA comparison comparison of of cell-free cell-free and and inin vivo vivo proteinprotein synthesis synthesis methods. methods. Through Through visualization visualization of theof themain main steps steps of in ofvitroin vitroand inand vivoin protein vivo protein expression, expression, the advantag the advantageses of cell-free of protein cell-free synthesis protein emerge.synthesis These emerge. include These the include eliminat theion elimination of the transformation of the transformation step, an step,open an reaction open reaction for direct for manipulationdirect manipulation of the environment of the environment of protein of proteinproduction production,, the lack theof constraints lack of constraints based on basedthe cell’s on life the objectives,cell’s life objectives, the channeling the channeling of all energy of all toward energy producti towardon production of the protein of the of protein interest, of and interest, the ability and the to storeability extracts to store extractsfor on-demand for on-demand protein protein expression. expression. Green Green cylinder cylinderss represent represent synthesized synthesized green green fluorescentfluorescent protein (GFP). While the number of cell-free platforms based on different organisms has grown substantially since itsits conception, conception, the the basic basic steps steps for successfulfor successfu implementationl implementation of a cell-free of a platformcell-free areplatform analogous are analogousacross platforms across (Figure platforms6). In (Figure brief, users6). In mustbrief, culture users must the cell culture line of the interest cell line from of interest which transcription from which transcriptionand translation and machinery translation are machinery to be extracted. are to be Next, extracted. the user Next, must the lyse user the must cells lyse while the maintaining cells while maintainingribosomal activity ribosomal in the activity lysate, preparein the lysate, cell extract prep byare clarifyingcell extract the by lysate clarifying through the various lysate methods,through and then utilize the prepared cell extract in CFPS reactions to synthesize the protein of interest. Methods Protoc. 2019, 2, 24 3 of 34 These basicMethods stepsand Protoc. have 2019, 2 many, 24 nuanced variations from platform to platform, and even3 of 38 within platforms. Lysis methods in particular are extremely variable and commonly used methods include homogenization,various methods, sonication, and then French utilize the press, prepared freeze cell thaw, extract nitrogen in CFPS reactions cavitation, to synthesize bead beating the protein [7]. Extract of interest. These basic steps have many nuanced variations from platform to platform, and even preparationwithin varies platforms. by centrifugation Lysis methods in speeds, particular run are off extremely reactions, variable dialysis, and orcommonly treatment used with methods nucleases to removeinclude endogenous homogenization, DNA or RNA.sonication, Here, French we report press, fr methodologieseeze thaw, nitrogen used cavitation, most commonly bead beating for [7]. obtaining highestExtract volumetric preparation yields varies of the by targetcentrifugation protein speeds, (Tables run1– 3off). Wereactions, also report dialysis, low or treatment adoption with platforms includingnucleases emerging to remove platforms
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