Oxalate improves protein synthesis by enhancing ATP supply in a cell-free system derived from Escherichia coli

The utilization efficiency of a secondary energy source in a cell-free protein synthesis system can be improved by use of a metabolic inhibitor. Oxalate, a potent inhibitor of phophoenolpyruvate synthetase, substantially increased the yield of chloramphenicol acetyltransferase synthesis through the enhanced supply of ATP. Oxalate, at 2.7 mM, increased the synthesis yield by 47% when successive amino acids additions prevent amino acid depletion during protein synthesis. These results suggest that cell-free protein synthesis efficiency could also be improved by disrupting the gene encoding phosphoenolpyruvate synthetase.     

Biosensor-assisted engineering of a high-yield Pichia pastoris cell-free protein synthesis platform

Cell-free protein synthesis (CFPS) has recently undergone a resurgence partly due to the proliferation of synthetic biology. The variety of hosts used for cell-free extract production has increased, which harnesses the diversity of cellular biosynthetic, protein folding, and posttranslational modification capabilities available. Here we describe a CFPS platform derived from Pichia pastoris, a popular recombinant protein expression host both in academia and the biopharmaceutical industry. A novel ribosome biosensor was developed to optimize the cell extract harvest time. Using this biosensor, we identified a potential bottleneck in ribosome content. Therefore, we undertook strain engineering to overexpress global regulators of ribosome biogenesis to increase in vitro protein production. CFPS extracts from the strain overexpressing FHL1 had a three-fold increase in recombinant protein yield compared with those from the wild-type X33 strain. Furthermore, our novel CFPS platform can produce complex therapeutic proteins, as exemplified by the production of human serum albumin to a final yield of 48.1 μg ml ⁻¹. Therefore, this study not only adds to the growing number of CFPS systems from diverse organisms but also provides a blueprint for rapidly engineering new strains with increased productivity in vitro that could be applied to other organisms.     

A rational approach to improving titer in Escherichia coli-based cell-free protein synthesis reactions

Cell-free protein synthesis (CFPS) is an established method for rapid recombinant protein production. Advantages like short synthesis times and an open reaction environment make CFPS a desirable platform for new and difficult-to-express products. Most recently, interest has grown in using the technology to make larger amounts of material. This has been driven through a variety of reasons from making site specific antibody drug conjugates, to emergency response, to the safe manufacture of toxic biological products. We therefore need robust methods to determine the appropriate reaction conditions for product expression in CFPS. Here we propose a process development strategy for Escherichia coli lysate-based CFPS reactions that can be completed in as little as 48 hr. We observed the most dramatic increases in titer were due to the E. coli strain for the cell extract. Therefore, we recommend identifying a high-producing cell extract for the product of interest as a first step. Next, we manipulated the plasmid concentration, amount of extract, temperature, concentrated reaction mix pH levels, and length of reaction. The influence of these process parameters on titer was evaluated through multivariate data analysis. The process parameters with the highest impact on titer were subsequently included in a design of experiments to determine the conditions that increased titer the most in the design space. This proposed process development strategy resulted in superfolder green fluorescent protein titers of 0.686 g/L, a 38% improvement on the standard operating conditions, and hepatitis B core antigen titers of 0.386 g/L, a 190% improvement.     

A highly efficient and economical cell-free protein synthesis system using the S12 extract of <Emphasis Type="Italic">Escherichia coli</Emphasis>

We have developed an economical and simple cell-free protein synthesis system that produces milligram quantities of proteins in a milliliter batch reaction. In this system, the S12 extract, which was prepared from glucose-adapted cells, was employed and glucose alone was successfully used for the efficient and stable regeneration of ATP. The ATP level in the reaction mixture remained stable over a remarkably extended reaction period, which enabled prolonged protein synthesis, and the issues associated with proton accumulation and amino acid depletion were simultaneously addressed. Under the reaction conditions established in this study, protein synthesis continued for 6 h and the amount of the accumulated protein reached 1.8 mg/mL.     

大肠杆菌细胞抽提物制备方案的简化与优化

无细胞蛋白表达系统是一种将目的蛋白在体外进行表达的新技术和新方法,已广泛应用到蛋白质组学、蛋白质结构和功能等领域的研究中。在无细胞蛋白表达系统中,细胞抽提物的制备是关键因素之一。通过对大肠杆菌细胞抽提物制备过程中离心速度、预孵化和透析等参数的考察,利用绿色荧光蛋白作为报告蛋白,可以得到一个细胞抽提物制备的简化方案。采用相对低的转速(12 000×g,10 min),简易空孵化即可制备出活性高的细胞抽提物,用于无细胞体系蛋白表达,其表达的绿色荧光蛋白产量为209μg/mL。与传统的大肠杆菌细胞抽提物S30相比较,新方案将使时间与成本节省62%,产量是传统方法的2.6倍,使无细胞蛋白表达技术的操作快速、高通量的优势更加明显。     

Increasing cell-free gene expression yields from linear templates in Escherichia coli and Vibrio natriegens extracts by using DNA-binding proteins

In crude extract-based cell-free protein synthesis (CFPS), DNA templates are transcribed and translated into functional proteins. Although linear expression templates (LETs) are less laborious and expensive to generate, plasmid templates are often desired over polymerase chain reaction-generated LETs due to increased stability and protection against exonucleases present in the extract of the reaction. Here we demonstrate that addition of a double stranded DNA-binding protein to the CFPS reaction, termed single-chain Cro protein (scCro), achieves terminal protection of LETs. This CroP-LET (scCro-based protection of LET) method effectively increases superfolder green fluorescent protein (sfGFP) expression levels from LETs in Escherichia coli CFPS reactions by sixfold. Our yields are comparable to other strategies that provide chemical and enzymatic DNA stabilization in E. coli CFPS. Notably, we also report that the CroP-LET method successfully enhanced yields in CFPS platforms derived from nonmodel organisms. Our results show that CroP-LET increased sfGFP yields by 18-fold in the Vibrio natriegens CFPS platform. With the fast-expanding applications of CFPS platforms, this method provides a practical and generalizable solution to protect linear expression DNA templates.     

Regeneration of adenosine triphosphate from glycolytic intermediates for cell-free protein synthesis.

A new approach for adenosine triphosphate (ATP) regeneration in a cell-free protein synthesis system is described. We first show that pyruvate can be used as a secondary energy source to replace or supplement the conventional secondary energy source, phosphoenol pyruvate (PEP). We also report that glucose-6-phosphate, an earlier intermediate of the glycolytic pathway, can be used for ATP regeneration. These new methods provide more stable maintenance of ATP concentration during protein synthesis. Because pyruvate and glucose-6-phosphate are the first and last intermediates of the glycolytic pathway, respectively, the results also suggest the possibility of using any glycolytic intermediate, or even glucose, for ATP regeneration in a cell-free protein synthesis system. As a result, the methods described provide cell-free protein synthesis with greater flexibility and cost efficiency.     

Energizing cell-free protein synthesis with glucose metabolism

In traditional cell-free protein synthesis reactions, the energy source (typically phosphoenolpyruvate (PEP) or creatine phosphate) is the most expensive substrate. However, for most biotechnology applications glucose is the preferred commercial substrate. Previous attempts to use glucose in cell-free protein synthesis reactions have been unsuccessful. We have now developed a cell-free protein synthesis reaction where PEP is replaced by either glucose or glucose-6-phosphate (G6P) as the energy source, thus allowing these reactions to compete more effectively with in vivo protein production technologies. We demonstrate high protein yields in a simple batch-format reaction through pH control and alleviation of phosphate limitation. G6P reactions can produce high protein levels ( approximately 700 microg/mL of chloramphenical acetyl transferase (CAT)) when pH is stabilized through replacement of the HEPES buffer with Bis-Tris. Protein synthesis with glucose as an energy source is also possible, and CAT yields of approximately 550 mug/mL are seen when both 10 mM phosphate is added to alleviate phosphate limitations and the Bis-Tris buffer concentration is increased to stabilize pH. By following radioactivity from [U-(14)C]-glucose, we find that glucose is primarily metabolized to the anaerobic products, acetate and lactate. The ability to use glucose as an energy source in cell-free reactions is important not only for inexpensive ATP generation during protein synthesis, but also as an example of how complex biological systems can be understood and exploited through cell-free biology.     

Scaling eukaryotic cell-free protein synthesis achieved with the versatile and high-yielding tobacco BY-2 cell lysate

Eukaryotic cell-free protein synthesis (CFPS) can accelerate expression and high-throughput analysis of complex proteins with functionally relevant post-translational modifications (PTMs). However, low yields and difficulties scaling such systems have prevented their widespread adoption in protein research and manufacturing. Here, we provide detailed demonstrations for the capabilities of a CFPS system derived from Nicotiana tabacum BY-2 cell culture (BY-2 lysate; BYL). BYL is able to express diverse, functional proteins at high yields in 48 h, complete with native disulfide bonds and N-glycosylation. An optimized version of the technology is commercialized as ALiCE® and advances in scaling of BYL production methodologies now allow scaling of eukaryotic CFPS reactions. We show linear, lossless scale-up of batch mode protein expression from 100 µL microtiter plates to 10 and 100 mL volumes in Erlenmeyer flasks, culminating in preliminary data from a litre-scale reaction in a rocking-type bioreactor. Together, scaling across a 20,000x range is achieved without impacting product yields. Production of multimeric virus-like particles from the BYL cytosolic fraction were then shown, followed by functional expression of multiple classes of complex, difficult-to-express proteins using the native microsomes of the BYL CFPS. Specifically: a dimeric enzyme; a monoclonal antibody; the SARS-CoV-2 receptor-binding domain; a human growth factor; and a G protein-coupled receptor membrane protein. Functional binding and activity are demonstrated, together with in-depth PTM characterization of purified proteins through disulfide bond and N-glycan analysis. Taken together, BYL is a promising end-to-end R&D to manufacturing platform with the potential to significantly reduce the time-to-market for high value proteins and biologics.     

The EnvZ protein of Salmonella typhimurium LT-2 and Escherichia coli K-12 is located in the cytoplasmic membrane

Abstract The osmoregulated expression of the porin proteins OmpC and OmpF in S. typhimurium and E. coli is dependent on the regulatory proteins OmpR and EnvZ. The function of the EnvZ protein is not clear. In order to establish the cellular location of EnvZ two different methods of buoyant sucrose density centrifugation was employed. The presence of EnvZ in the different fractions was visualised by immunoblotting. It was conclusively shown that the EnvZ protein is located in the cytoplasmic membrane fraction. The result is in agreement with the available sequence data which shows that the EnvZ polypeptide contains two long hydrophobic stretches.     

Prolonged cell-free protein synthesis using dual energy sources: Combined use of creatine phosphate and glucose for the efficient supply of ATP and retarded accumulation of phosphate

The accumulation of inorganic phosphate inhibits protein synthesis in cell-free protein synthesis reactions that are energized by high-energy-phosphate-containing compounds. This study developed a new scheme for supplying energy using dual energy sources to enhance the regeneration of ATP and lower the rate of phosphate accumulation. In the proposed scheme, where creatine phosphate (CP) and glucose were simultaneously used as the energy sources, the phosphate released from the CP was subsequently used in the glycolytic pathway for the utilization of the glucose, which enhanced the ATP supply and reduced the rate of inorganic phosphate accumulation. When tested against different proteins, the developed method produced 2-3 times more protein than the conventional ATP regeneration methods using single energy sources.     

Bacterial cell-free system for high-throughput protein expression and a comparative analysis of Escherichia coli cell-free and whole cell expression systems

Sixty-three proteins of Pseudomonas aeruginosa in the size range of 18-159 kDa were tested for expression in a bacterial cell-free system. Fifty-one of the 63 proteins could be expressed and partially purified under denaturing conditions. Most of the expressed proteins showed yields greater than 500 ng after a single affinity purification step from 50 microl in vitro protein synthesis reactions. The in vitro protein expression plus purification in a 96-well format and analysis of the proteins by SDS-PAGE were performed by one person in 4 h. A comparison of in vitro and in vivo expression suggests that despite lower yields and less pure protein preparations, bacterial in vitro protein expression coupled with single-step affinity purification offers a rapid, efficient alternative for the high-throughput screening of clones for protein expression and solubility.     

Transport of lactate and acetate through the energized cytoplasmic membrane of Escherichia coli

Escherichia coli produces lactate and acetate in significant amounts during both aerobic and anaerobic glycolysis. A model describing the mechanism of protein mediated lactate transport has previously bee proposed. A simple theoretical analysis here indicates that the proposed model would be drain cellular energy resources by catalytically dissipating the proton-motive force. An experimental analysis of lactate and acetate transport employ nuclear magnetic resonance (NMR) spectroscopy to measure the relative concentration of these end products on the two sides of the cytoplasmic membrane of anaerobically glycolyzing cells. Comparison of measured concentration rations to those expected at equilibrium for various transport modes indicates that acetate is a classical uncoupling agent, permeating the membrane oat comparable rates in the dissociated and undissociated forms. The lactate concentration ratio changes market markedly after an initial period of sustained glycolysis. This change is most readily explained as resulting from a lactate transport system that responds to an indicator of glycolytic activity. The data further indicates that lactate permeates the membrane in both dissociated and undissociated forms. Both acids, then are capable of catalytically dissipating the proton-motives force. (c) 1995 John Wiley & Sons, Inc.     

无细胞蛋白表达体系研究进展及在生物制药领域中的应用

作为一种快速高效的体外蛋白合成手段,无细胞蛋白表达体系(Cell-free Protein Synthesis,CFPS)一直以来就被广泛应用于基础生物学领域的研究。与传统的基于细胞的体内表达体系相比,CFPS突破了细胞的生理限制,其可调控性强、对毒性蛋白的耐受力高,使得许多很难在体内合成的复杂蛋白在体外顺利表达。近年来随着研究人员不断对CFPS进行优化,通过简化制备工艺、开发价格低廉的能量再生系统、稳定底物供应、促进蛋白正确折叠等方式,成功研发出生产效率高、成本低廉、反应体积大的表达体系。凭借其高通量和大规模的蛋白表达优势,CFPS为解决生物制药领域中面临的难题提供了新的解决思路,并成功地应用于高通量药物筛选、大规模生产重组蛋白药物、个体化定制肿瘤疫苗等领域,显示出其在生物制药领域的重要应用潜力。     

Amino acid balancing for the prediction and evaluation of protein concentrations in cell-free protein synthesis systems

Cell-free protein synthesis (CFPS) systems are an attractive method to complement the usual cell-based synthesis of proteins, especially for screening approaches. The literature describes a wide variety of CFPS systems, but their performance is difficult to compare since the reaction components are often used at different concentrations. Therefore, we have developed a calculation tool based on amino acid balancing to evaluate the performance of CFPS by determining the fractional yield as the ratio between theoretically achievable and experimentally achieved protein molar concentration. This tool was applied to a series of experiments from our lab and to various systems described in the literature to identify systems that synthesize proteins very efficiently and those that still have potential for higher yields. The well-established Escherichia coli system showed a high efficiency in the utilization of amino acids, but interestingly, less considered systems, such as those based on Vibrio natriegens or Leishmania tarentolae, also showed exceptional fractional yields of over 70% and 90%, respectively, implying very efficient conversions of amino acids. The methods and tools described here can quickly identify when a system has reached its maximum or has limitations. We believe that this approach will facilitate the evaluation and optimization of existing CFPS systems and provides the basis for the systematic development of new CFPS systems.     

Effect of energy source on the efficiency of translational termination during cell-free protein synthesis

We studied how the fidelity of translation termination is affected by the method of ATP regeneration during cell-free protein synthesis. During the in vivo expression of hEPO, whose termination is directed by the UGA codon, we found that substantial proportions of the translational products showed a larger molecular weight than expected. Similar results were obtained in a cell-free synthesis reaction using phosphoenol pyruvate (PEP) or 3-phosphoglycerate (3PG) for ATP regeneration. However, when the energy source was switched to creatine phosphate (CP), the readthrough of the UGA codon was completely repressed and only the target protein of the correct size was expressed in a high yield. To the best of our knowledge, this is the first report describing the relationship between the regeneration of nucleotide triphosphates and protein readthrough, and we also believe that the discovery would pave the way to the selective and efficient expression of target proteins in cell-free protein synthesis systems.