Comparison of cell‐based and cell‐free protocols for producing target proteins from the<i>Arabidopsis thaliana</i>genome for structural studies

Tyler, Robert C.; Aceti, David J.; Bingman, C.A.; Cornilescu, Claudia C.; Fox, Brian G.; Frederick, Ronnie O.; Jeon, Won Bae; Lee, Min S.; Newman, Christopher; Peterson, Francis C.; Phillips, George N.; Shahan, Mark N.; Singh, Shanteri; Song, Jikui; Sreenath, Hassan K.; Tyler, Ejan M.; Ulrich, Eldon L.; Vinarov, Dmitriy A.; Vojtik, Frank C.; Volkman, Brian F.; Wrobel, Russell L.; Zhao, Qin; Markley, John L.

doi:10.1002/prot.20436

Cited by 57 publications

(48 citation statements)

References 21 publications

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“…2) modified by the Center of Eukaryotic Structural Genomics (24 -26). For expression in E. coli, the dwarf sperm whale, gray seal, goosebeak whale, and sperm whale Mb genes were cloned into the pVP80K vector, and sequence-specific ribosome-binding sites were designed utilizing the Salis Lab Calculator to optimize yields (27,28). Myoglobin mutants were constructed in these vectors using PIPE site-directed mutagenesis (29).…”

Section: Methodsmentioning

confidence: 99%

Apoglobin Stability Is the Major Factor Governing both Cell-free and in Vivo Expression of Holomyoglobin

Samuel

Smith

Phillips

et al. 2015

Journal of Biological Chemistry

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Background: Myoglobin expression is highly variable in animal tissues and in E. coli. Results: The parameters governing holomyoglobin expression were determined experimentally. Conclusion: Both cell-free and in vivo holomyoglobin yields depend quantitatively on the stability of apoglobin and not hemin affinity. Significance: Enhanced apomyoglobin stability is required for high levels of holoprotein expression due to rapid rates of aggregation of unfolded apoproteins.

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Section: Methodsmentioning

confidence: 99%

Apoglobin Stability Is the Major Factor Governing both Cell-free and in Vivo Expression of Holomyoglobin

Samuel

Smith

Phillips

et al. 2015

Journal of Biological Chemistry

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“…Certainly, this has been shown for soluble proteins (Yokoyama 2003;Tyler et al 2005), but, with the importance of translocation and proper folding within the membrane, this is not so obvious for MPs. Much of the initial CF work on MPs, however, was showing activity for channels and transporters (Berrier et al 2004;Elbaz et al 2004).…”

Section: Cell-free Proteins Behave Like Their In Vivo Counterpartsmentioning

confidence: 99%

“…These adaptations include coupled transcription and translation, enzymatic subsystems to regenerate high-energy nucleotides, and continuousexchange ''feeding'' systems via dialysis, all of which allow the reaction to proceed at higher levels for a longer amount of time. Successful CF systems, both prokaryotic (Kigawa et al 1999;Klammt et al 2004) and eukaryotic (Endo and Sawasaki 2003;Tyler et al 2005), have been described. Furthermore, CF-expressed MPs have also recently been used in nuclear magnetic resonance (NMR) experiments (Koglin et al 2006).…”

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confidence: 99%

Cell‐free complements in vivo expression of the E. coli membrane proteome

et al. 2007

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Reconstituted cell-free (CF) protein expression systems hold the promise of overcoming the traditional barriers associated with in vivo systems. This is particularly true for membrane proteins, which are often cytotoxic and due to the nature of the membrane, difficult to work with. To evaluate the potential of cellfree expression, we cloned 120 membrane proteins from E. coli and compared their expression profiles in both an E. coli in vivo system and an E. coli-derived cell-free system. Our results indicate CF is a more robust system and we were able to express 63% of the targets in CF, compared to 44% in vivo. To benchmark the quality of CF produced protein, five target membrane proteins were purified and their homogeneity assayed by gel filtration chromatography. Finally, to demonstrate the ease of amino acid labeling with CF, a novel membrane protein was substituted with selenomethionine, purified, and shown to have 100% incorporation of the unnatural amino acid. We conclude that CF is a novel, robust expression system capable of expressing more proteins than an in vivo system and suitable for production of membrane proteins at the milligram level.Keywords: cell-free protein expression; integral membrane proteins; structural genomics; high-throughput protein expression Integral membrane proteins (MPs), despite their biological importance, currently account for <1% of all known high resolution protein structures. MPs are notoriously difficult to work with, and expression, detergent solubilization, purification, and crystallization all present unique challenges over their soluble counterparts (White 2004). MPs generally express at much lower levels than soluble proteins and, when in vivo overexpression is successful, the protein can be cytotoxic or incorporated into insoluble inclusion bodies. Following successful MP expression, a suitable detergent condition must also be found that simultaneously extracts the protein from the membrane while retaining the native fold and function. This protein-detergent complex (PDC) is often heterogeneous, creating numerous problems in purification and crystallization. Optimizing purification, assaying protein function, and crystallization all require milligram quantities of protein, and MP expression is therefore a limiting step in macromolecular structure determination (Dobrovetsky et al. 2005;Eshaghi et al. 2005;Korepanova et al. 2005;Columbus et al. 2006;Surade et al. 2006). One recognized alternative is cell-free (CF) expression (Klammt et al. 2004). 4 These authors contributed equally to this work.

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“…These two systems were also tested on a Plasmodium falciparum protein set and while detectable protein was obtained for 30% of the proteins in E. coli, protein was obtained for 75% when expressed in the eukaryotic cell-free system (Tyler et al, 2005). Thus, eukaryotic in vitro systems, specifically the wheat germ cell-free system, hold significant promise.…”

Section: Protein Expressionmentioning

confidence: 99%

Wheat germ cell-free expression system as a pathway to improve protein yield and solubility for the SSGCID pipeline

et al. 2011

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Recombinant expression of proteins of interest in Escherichia coli is an important tool in the determination of protein structure. However, lack of expression and insolubility remain significant challenges to the expression and crystallization of these proteins. The SSGCID program uses a wheat germ cellfree expression system as a rescue pathway for proteins that are either not expressed or insoluble when produced in E. coli. Testing indicates that the system is a valuable tool for these protein targets. Further increases in solubility were obtained by the addition of the NVoy polymer reagent to the reaction mixture. These data indicate that this eukaryotic cell-free expression system has a high success rate and that the addition of specific reagents can increase the yield of soluble protein.

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Comparison of cell‐based and cell‐free protocols for producing target proteins from theArabidopsis thalianagenome for structural studies

Cited by 57 publications

References 21 publications

Apoglobin Stability Is the Major Factor Governing both Cell-free and in Vivo Expression of Holomyoglobin

Apoglobin Stability Is the Major Factor Governing both Cell-free and in Vivo Expression of Holomyoglobin

Cell‐free complements in vivo expression of the E. coli membrane proteome

Wheat germ cell-free expression system as a pathway to improve protein yield and solubility for the SSGCID pipeline

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