Rational improvement of cell-free protein synthesis

Pedersen, Anders; Hellberg, Kristofer; Enberg, Johan; Karlsson, Bengt

doi:10.1016/j.nbt.2010.06.015

Cited by 45 publications

(49 citation statements)

References 20 publications

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“…S2). The dispensability of amino acids and rNTPs was not unexpected because a previous study reported that glutamine and serine were sufficient for cell-free protein expression using S30 fractions [21].…”

Section: Resultsmentioning

confidence: 96%

“…The expression levels of sfGFP were estimated by measuring the fluorescence intensity by SDS-PAGE performed with unboiled samples. As a control, S30 lysates containing buffer and salts (Buf-S30) were prepared; in these lysates, typical S30 buffer was used instead of DDW [20], [21]. The S30 fractions were mixed with reaction mixtures for cell-free expression and plasmid DNA encoding sfGFP and incubated at 37°C.…”

Section: Resultsmentioning

confidence: 99%

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Condensation of an Additive-Free Cell Extract to Mimic the Conditions of Live Cells

Fujiwara

Nomura

2013

PLoS ONE

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The cellular environment differs from that of reconstituted materials mainly because of the presence of highly condensed biomacromolecules. To mimic the environment and conditions in living cells, we developed a method to prepare additive-free, highly concentrated cell extracts. First, we verified the requirement for specific salts and buffers for functional cell-free translation extracts. The S30 fraction of Escherichia coli cell extracts without additives exhibited sufficient cell-free protein production. Next, we established a method to accumulate biological components by gradual evaporation by using a vacuum desiccator. Bovine serum albumin, green fluorescent protein, alkaline phosphatase, and a diluted reconstituted protein expression system were successfully condensed in their active forms using this method. The protein concentration of the prepared cell extract was elevated to 180 mg/mL, which was expected to contain approximately 260 mg/mL macromolecules, without the loss of cell-free protein expression activity. Such a condensed cell extract may be useful for investigating the differences between cells and reconstituted materials and may contribute to the development of methods to synthesize cells from cell extracts in the future.

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Section: Resultsmentioning

confidence: 96%

Section: Resultsmentioning

confidence: 99%

Condensation of an Additive-Free Cell Extract to Mimic the Conditions of Live Cells

Fujiwara

Nomura

2013

PLoS ONE

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“…Intein self-cleavage was induced by flushing the column with buffer containing 40 mM DTT followed by incubation for 48 h. Bax was further purified by anion exchange chromatography using a mono-Q column (GE Healthcare) and size exclusion chromatography using a HiPrep 16/60 Sephacryl S-100 HR column (GE Healthcare). Cell-free synthesis of the full-length Bcl-2 was performed as previously described [24], [25], but with the addition of 0.1% w/v Brij-35 instead of polyoxyethylene-(20)-cetyl-ether (Brij-58) during the expression. For the subsequent purification steps 0.05% w/v Brij-35 was used.…”

Section: Methodsmentioning

confidence: 99%

Reconstitution of the Anti-Apoptotic Bcl-2 Protein into Lipid Membranes and Biophysical Evidence for Its Detergent-Driven Association with the Pro-Apoptotic Bax Protein

et al. 2013

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The anti-apoptotic B-cell CLL/lymphoma-2 (Bcl-2) protein and its counterpart, the pro-apoptotic Bcl-2-associated X protein (Bax), are key players in the regulation of the mitochondrial pathway of apoptosis. However, how they interact at the mitochondrial outer membrane (MOM) and there determine whether the cell will live or be sentenced to death remains unknown. Competing models have been presented that describe how Bcl-2 inhibits the cell-killing activity of Bax, which is common in treatment-resistant tumors where Bcl-2 is overexpressed. Some studies suggest that Bcl-2 binds directly to and sequesters Bax, while others suggest an indirect process whereby Bcl-2 blocks BH3-only proteins and prevents them from activating Bax. Here we present the results of a biophysical study in which we investigated the putative interaction of solubilized full-length human Bcl-2 with Bax and the scope for incorporating the former into a native-like lipid environment. Far-UV circular dichroism (CD) spectroscopy was used to detect direct Bcl-2-Bax-interactions in the presence of polyoxyethylene-(23)-lauryl-ether (Brij-35) detergent at a level below its critical micelle concentration (CMC). Additional surface plasmon resonance (SPR) measurements confirmed this observation and revealed a high affinity between the Bax and Bcl-2 proteins. Upon formation of this protein-protein complex, Bax also prevented the binding of antimycin A2 (a known inhibitory ligand of Bcl-2) to the Bcl-2 protein, as fluorescence spectroscopy experiments showed. In addition, Bcl-2 was able to form mixed micelles with Triton X-100 solubilized neutral phospholipids in the presence of high concentrations of Brij-35 (above its CMC). Following detergent removal, the integral membrane protein was found to have been fully reconstituted into a native-like membrane environment, as confirmed by ultracentrifugation and subsequent SDS-PAGE experiments.

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“…Thus, protein synthesis is disconnected from cell fate and the protein synthesis reaction itself is not constrained by a cell wall, meaning that reaction conditions are accessible from the outside of the reaction vessel [5,7,42].…”

Section: Cell-free Protein Synthesismentioning

confidence: 99%

Cell-Free Synthesis Meets Antibody Production: A Review

Stech

Kubick

2015

Antibodies

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Engineered antibodies are key players in therapy, diagnostics and research. In addition to full size immunoglobulin gamma (IgG) molecules, smaller formats of recombinant antibodies, such as single-chain variable fragments (scFv) and antigen binding fragments (Fab), have emerged as promising alternatives since they possess different advantageous properties. Cell-based production technologies of antibodies and antibody fragments are well-established, allowing researchers to design and manufacture highly specific molecular recognition tools. However, as these technologies are accompanied by the drawbacks of being rather time-consuming and cost-intensive, efficient and powerful cell-free protein synthesis systems have been developed over the last decade as alternatives. So far, prokaryotic cell-free systems have been the focus of interest. Recently, eukaryotic in vitro translation systems have enriched the antibody production pipeline, as these systems are able to mimic the natural pathway of antibody synthesis in eukaryotic cells. This review aims to overview and summarize the advances made in the production of antibodies and antibody fragments in cell-free systems.

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Rational improvement of cell-free protein synthesis

Cited by 45 publications

References 20 publications

Condensation of an Additive-Free Cell Extract to Mimic the Conditions of Live Cells

Condensation of an Additive-Free Cell Extract to Mimic the Conditions of Live Cells

Reconstitution of the Anti-Apoptotic Bcl-2 Protein into Lipid Membranes and Biophysical Evidence for Its Detergent-Driven Association with the Pro-Apoptotic Bax Protein

Cell-Free Synthesis Meets Antibody Production: A Review

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