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

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 cell-free 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.     

Alternative fermentation conditions for improved Escherichia coli-based cell-free protein synthesis for proteins requiring supplemental components for proper synthesis

Escherichia coli-based cell-free protein synthesis is a powerful emerging tool for protein engineering due to the open, accessible nature of the reaction and its straightforward, economical potential for many diverse applications. One critical limitation of this system is the inability to express some complex, eukaryotic, and/or unnatural proteins at high expression yields. A potential solution is a synthetic-biology-like approach where cell-free reactions are supplemented by expressing the required supplemental components in the E. coli cells during the fermentation, which cells are used to prepare the extract for cell-free protein synthesis. Here we report adjustments to the fermentation conditions that increase yields of complex proteins upwards of 150% over standard conditions. We consider extracts containing GroEL/ES protein folding chaperones and extracts containing orthogonal tRNA/tRNA synthetase pairs for noncanonical amino acid incorporation. In contrast to standard cell-free synthesis, delaying the harvest of supplemented fermentations lead to increased and more consistent yields of proteins that required supplemental components. Protein yields enhanced by buffering the fermentation media pH lead to an average 52% decrease in yield cost, while costs for cases unchanged or negatively affected by buffering increased an average 14%. An apparent balance is required between the supplemental components and general extract protein profile.     

Expression of G protein coupled receptors in a cell-free translational system using detergents and thioredoxin-fusion vectors

In Escherichia coli and other cell-based expression systems, there are critical difficulties in synthesizing membrane proteins, such as the low protein expression levels and the formation of insoluble aggregates. However, structure determinations by X-ray crystallography require the purification of milligram quantities of membrane proteins. In this study, we tried to solve these problems by using cell-free protein expression with an E. coli S30 extract, with G protein coupled receptors (GPCRs) as the target integral membrane proteins. In this system, the thioredoxin-fusion vector induced high protein expression levels as compared with the non-fusion and hexa-histidine-tagged proteins. Two detergents, Brij35 and digitonin, effectively solubilized the produced GPCRs, with little or no effect on the protein yields. The synthesized proteins were detected by Coomassie brilliant blue staining within 1h of reaction initiation, and were easily reconstituted within phospholipid vesicles. Surprisingly, the unpurified, reconstituted thioredoxin-fused receptor proteins had functional activity, in that a specific affinity binding value of an antagonist was obtained for the receptor. This cell-free translation system (about 1mg/ml of reaction volume for 6-8 h) has biophysical and biochemical advantages for the synthesis of integral membrane proteins.     

Development of cell-free protein synthesis platforms for disulfide bonded proteins

The use of cell-free protein synthesis (CFPS) for recombinant protein production is emerging as an important technology. For example, the openness of the cell-free system allows control of the reaction environment to promote folding of disulfide bonded proteins in a rapid and economically feasible format. These advantages make cell-free protein expression systems particularly well suited for producing patient specific therapeutic vaccines or antidotes in response to threats from natural and man-made biological agents and for pharmaceutical proteins that are difficult to produce in living cells. In this work we assess the versatility of modern cell-free methods, optimize expression and folding parameters, and highlight the importance of rationally designed plasmid templates for producing mammalian secreted proteins, fusion proteins, and antibody fragments in our E. coli-based CFPS system. Two unique CFPS platforms were established by developing standardized extract preparation protocols and generic cell-free reaction conditions. Generic reaction conditions enabled all proteins to express well with the best therapeutic protein yield at 710 microg/mL, an antibody fragment at 230 microg/mL, and a vaccine fusion protein at 300 microg/mL; with the majority correctly folded. Better yields were obtained when cell-free reaction conditions were optimized for each protein. Establishing general CFPS platforms enhances the potential for cell-free protein synthesis to reliably produce complex protein products at low production and capital costs with very rapid process development timelines.     

Enhanced in vitro translation at reduced temperatures using a cold-shock RNA motif

Cell-free synthesis of recombinant proteins has emerged as an alternative method of protein production although protein yields still cannot compete with in vivo expression techniques. In systems based on S30 extracts of Escherichia coli unfavorable side-reactions are involved in limiting protein yields. Therefore, carrying out cell-free reactions at lower temperatures might be beneficial as side reactions should be decreased. In this study we show that by using the 5′-untranslated region of the cold-shock gene cspA from E. coli as mRNA leader in cell-free reactions, the expression temperature can be decreased and simultaneously leads to an increase in protein yields. A compensation for the lower activity of T7 RNA polymerase at lower temperatures enhances protein synthesis even further. Additionally, this 5′-untranslated region also standardizes the optimal expression temperature of different proteins.     

High-yield cell-free protein synthesis for site-specific incorporation of unnatural amino acids at two sites

Using aminoacyl-tRNA synthetase/suppressor tRNA pairs derived from Methanocaldococcus jannaschii, an Escherichia coli cell-free protein production system affords proteins with site-specifically incorporated unnatural amino acids (UAAs) in high yields through the use of optimized amber suppressor tRNA(CUA)(opt) and optimization of reagent concentrations. The efficiency of the cell-free system allows the incorporation of trifluoromethyl-phenylalanine using a polyspecific synthetase evolved previously for p-cyano-phenylalanine, and the incorporation of UAAs at two different sites of the same protein without any re-engineering of the E. coli cells used to make the cell-free extract.     

High-level expression of human beta-defensin-2 gene with rare codons in E. coli cell-free system

Human beta-defensin-2 (hBD2) is A small cationic peptide with A broad range of antimicrobial activity. An E. coli cell-free system was employed to express the hBD2 fusion protein by using the hBD2 gene with 14 rare codons. The results showed that the expression level of trxA-hBD2 fusion protein was 0.35 mg/ml, which is the same as that obtained with A synthetic codon-optimized gene. By using another fusion partner (GFP), similar high-level expression was also achieved in this cell-free system. This meant that human beta-defensin-2 gene could be directly used to express hBD2 fusion protein efficiently in an E. coli cell-free system without the optimization of codons. The expression level of hBD2 fused with thioredoxin could be further improved up to 2.0 mg/ml by adopting A continuous exchange cell-free system. A simple one-stage affinity purification procedure was also developed to recover this fusion protein efficiently.     

Cell-free expression of disulfide-containing eukaryotic proteins for structural biology

We describe Escherichia?coli based cell-free production of milligram quantities of eukaryotic proteins containing native disulfide bonds. Using a previously described expression system, we systematically investigated the influence of redox potential variation in the reaction mixture and the impact of adding disulfide bond catalysts on soluble protein production. It is then shown that the optimized reaction conditions for native disulfide bond formation can be combined with the use of N-terminal fusion constructs with the GB1 domain for increased expression yields. The resulting cell-free system is suitable for stable-isotope labeling and does not require chemical pretreatment of the cell extract to stabilize the redox potential. For the human doppel protein, the mouse doppel protein and mouse interleukin-22 we obtained 0.3-0.7?mg of purified native protein per milliliter of reaction mixture. Formation of disulfide bonds was validated using the Ellman assay, and native folding of the three proteins was monitored by NMR and CD spectroscopy. Structured digital abstract ? mIL22?and?mIL22?bind?by?nuclear magnetic resonance?(View interaction).     

High-level cell-free synthesis yields of proteins containing site-specific non-natural amino acids

We describe an E. coli-based cell-free system for the production of proteins with a non-natural amino acid (nnAA) incorporated site-specifically (modified protein). The mutant Methanococcus jannaschii tyrosyl-tRNA synthetase (mTyrRS) and tRNA(Tyr) pair were used as orthogonal elements. The mTyrRS experienced proteolysis and modified protein yields improved with higher synthetase addition (200-300 microg/mL). Product yields were also improved by increasing levels of total protein to 20 mg protein/mL and available vesicle surface area to 0.5 m(2)/mL. This new E. coli-based cell-free procedure produced up to 400 microg/mL of eCAT109pAz, 660 microg/mL of eDHFR10pAz, and 210 microg/mL of mDHFR31pAz with p-azido-L-phenylalanine (pAz) incorporated site-specifically at the amber nonsense codon. O-methyl-L-tyrosine and p-acetyl-L-phenylalanine were incorporated by similar protocols. The desired specificity for incorporation of the nnAA by the cell-free system was confirmed. Additionally, the modified proteins were enzymatically active and reactive for copper(I)-catalyzed (3 + 2) cycloadditions (click chemistry).     

A Continuous-Exchange Cell-Free Protein Synthesis System Based on Extracts from Cultured Insect Cells

In this study, we present a novel technique for the synthesis of complex prokaryotic and eukaryotic proteins by using a continuous-exchange cell-free (CECF) protein synthesis system based on extracts from cultured insect cells. Our approach consists of two basic elements: First, protein synthesis is performed in insect cell lysates which harbor endogenous microsomal vesicles, enabling a translocation of de novo synthesized target proteins into the lumen of the insect vesicles or, in the case of membrane proteins, their embedding into a natural membrane scaffold. Second, cell-free reactions are performed in a two chamber dialysis device for 48 h. The combination of the eukaryotic cell-free translation system based on insect cell extracts and the CECF translation system results in significantly prolonged reaction life times and increased protein yields compared to conventional batch reactions. In this context, we demonstrate the synthesis of various representative model proteins, among them cytosolic proteins, pharmacological relevant membrane proteins and glycosylated proteins in an endotoxin-free environment. Furthermore, the cell-free system used in this study is well-suited for the synthesis of biologically active tissue-type-plasminogen activator, a complex eukaryotic protein harboring multiple disulfide bonds.     

Enhanced cell-free protein expression by fusion with immunoglobulin Ckappa domain

While cell-free systems are increasingly used for protein expression in structural and functional studies, several proteins are difficult to express or expressed only at low levels in cell-free lysates. Here, we report that fusion of the human immunoglobulin kappa light chain constant domain (Ckappa) at the C terminus of four representative proteins dramatically improved their production in the Escherichia coli S30 system, suggesting that enhancement of cell-free protein expression by Ckappa fusion will be widely applicable.     

Application of the wheat-germ cell-free translation system to produce high temperature requirement A3 (HtrA3) proteases

Mammalian high temperature requirement A3 (HtrA3) is a serine protease of the HtrA family. It is an important factor for placental development and a tumor suppressor. The biochemical properties of HtrA3 are uncharacterized. One critical step in biochemical characterization is overexpressing and purifying the full-length recombinant protein. However, utility of cell-based expression systems is limited for a protease because of autocleavage. The wheat-germ cell-free translation system is highly efficient at producing "difficult" eukaryotic multidomain proteins and is easily modifiable for protein synthesis at different temperatures. In this study, we evaluated the potential of the wheat-germ cell-free translation system for producing human HtrA3. HtrA3 underwent autocleavage when synthesized at 17 °C. When the synthesis temperature was lowered to 4 °C, full-length HtrA3 was successfully produced and proteolytically active. Catalytic site serine substitution with alanine (S305A) stabilized HtrA3 while abolishing its protease activity. This mutant was readily synthesized and stable at 17 °C. When used with glutathione S-transferase (GST) pull-down assay, S305A HtrA3 was a valuable bait in searching for endogenous HtrA3 binding proteins. Thus, we demonstrated the unique utility of the wheat-germ cell-free translation system for producing and characterizing human HtrA3. These strategies will be likely applicable to a wide range of proteases.     

High-throughput,genome-scale protein production method based on the wheat germ cell-free expression system

Current cell-free protein expression systems are capable of synthesizing proteins with high speed and accuracy; however, the yields are low due to their instability over time. Escherichia coli based systems are not always sufficient for expression of eukaryotic proteins. This report reviews a high-throughput protein production method based on the cell-free system prepared from eukaryote, wheat embryos. We first demonstrate a method for preparation of this extract that exhibited a high degree of stability and activity. To maximize translation yield and throughput, we address and resolve the following issues: (1) optimization of the ORF flanking regions; (2) PCR-based generation of DNA for mRNA production; (3) expression vectors for large-scale protein production; and (4) a translation reaction that does not require a membrane. The combination of these elemental processes with robotic automation resulted in high-throughput protein synthesis.     

Increasing PCR fragment stability and protein yields in a cell-free system with genetically modified Escherichia coli extracts

Escherichia coli cell-free protein synthesis is a highly productive system that can be applied to high throughput expression from polymerase chain reaction (PCR) products in 96-well plates for proteomic studies as well as protein evolution. However, linear DNA instability appears to be a major limitation of the system. We modified the genome of the E. coli strain A19 by removing the endA gene encoding the endonuclease I and replacing the recCBD operon (in which recD encodes the exonuclease V) by the lambda phage recombination system. Using the cell extract from this new strain increased the stability of PCR products amplified from a plasmid containing the cat gene. This resulted in CAT (chloramphenicol acetyltransferase) production from PCR products comparable to that from plasmids (500-600 microg/ml) in a batch reaction. We show that cell-free protein synthesis reactions using PCR products amplified from genomic DNA and extended with the T7 promoter and the T7 terminator give the same high yields of proteins (550 microg/ml) in 96-well plates. With this system, it was possible to rapidly express a range of cytoplasmic and periplasmic proteins.     

Characterization of human papillomavirus type 11 E1 and E2 proteins expressed in insect cells.

The study of human papillomavirus replication has been hampered by the lack of an in vitro system which reliably supports virus replication. Recent results from the bovine papillomavirus (BPV) system indicate that the E1 and E2 proteins are the only viral gene products required for replication. By analogy with simian virus 40 large T antigen, E1 is thought to possess ATPase and helicase activity, which may play a direct role in viral DNA replication. The precise role of E2 is unclear, but it may function in part to help localize E1 to the replication origin. We have initiated a study of replication in the human papillomavirus type 11 system which, by analogy to BPV, has focused on the E1 and E2 proteins of this virus. We have expressed the full-length E1 and E2 proteins in Sf9 insect cells by using a baculovirus expression vector. Both the 80-kDa E1 protein and the 42.5-kDa E2 protein are nuclear phosphoproteins. The E1 and E2 proteins form a heteromeric complex within the insect cells, and both proteins localize to a DNA fragment which contains the viral origin of replication. In addition, we have detected an E1-associated ATPase and GTPase activity, which is likely part of an energy-generating system for the helicase activity which is predicted for this protein. The human papillomavirus type 11 E1 and E2 proteins possess the same replication-associated activities exhibited by the corresponding BPV proteins, suggesting that the replication activities of these viruses are tightly conserved.     

Crystallization of the membrane protein hVDAC1 produced in cell-free system

Structural studies of membrane proteins are in constant evolution with the development of new improvements for their expression, purification, stabilization and crystallization. However, none of these methods still provides a universal approach to solve the structure of membrane proteins. Here we describe the crystallization of the human voltage-dependent anion channel-1 produced by a bacterial cell-free expression system. While VDAC structures have been recently solved, we propose an alternative strategy for producing the recombinant protein, which can be applied to other membrane proteins reluctant to expression, purification and crystallization by classical approaches. Despite a lot of efforts to crystallize a cell-free expressed membrane protein, this study is to our knowledge one of the first reports of a successful crystallization. Focusing on expression in a soluble and functional state, in a detergent environment, is the key to get crystals. Although the diffraction of VDAC crystals is limited, the simplicity and the rapidity to set-up and optimize this technology are drastic advantages in comparison to other methods.