102 Biotechnol. Prog. 2004, 20, 102−109 Rapid Expression and Purification of 100 nmol Quantities of Active Protein Using Cell-Free Protein Synthesis Michael C. Jewett and James R. Swartz* Department of Chemical Engineering, Stanford University, Stanford, California 94305-5025 Two strategies for ATP regeneration during cell-free protein synthesis were applied to the large-scale production and single-column purification of active chloramphenicol acetyl transferase (CAT). Fed-batch reactions were performed on a 5-10 mL scale, approximately 2 orders of magnitude greater than the typical reaction volume. The pyruvate oxidase system produced 104 nmol of active CAT ina5mLreaction over the course of 5 h. The PANOx system produced 261 ( 42 nmol, about 7 mg, of active CAT in a 10 mL reaction over the course of 4 h. The reaction product was purified to apparent homogeneity with approximately 70% yield by a simple affinity chromatog- raphy adsorption and elution. To our knowledge, this is the largest amount of actively expressed protein to be reported in a simple, fed-batch cell-free protein synthesis reaction. Introduction enous acetate kinase present in the cell extract prepared from E. coli. The most notable feature of this system is Cell-free protein synthesis offers a method to exploit that phosphate released during protein synthesis is biological processes without intact cells. It is a convenient recycled into acetyl-phosphate (Figure 1A). This attribute tool for the production of active recombinant DNA (rDNA) allows for numerous additions of the energy source proteins and has several advantages over in vivo protein without inhibition of the protein synthesis reaction via expression systems (1-4). The productivity of cell-free systems has increased more than 100 times over the past phosphate accumulation. Previous attempts to scale-up decade, and this technology is becoming more than just the protein synthesis reaction using the pyruvate oxidase a laboratory research technique. The surge in productiv- energy regeneration system were unsuccessful. It was ity of cell-free systems is founded on a new understanding reported that scaling up a batch reaction from 15 to 120 of energy source limitations present in cell-free reactions. µL decreased the protein synthesis yield by more than However, three main challenges still remain for a broader 60% (9). Most likely, the simple batch configuration acceptance of cell-free protein synthesis as a general utilized for these reactions was limited by transfer of method for the production of rDNA proteins. One of these oxygen into the reaction solution. Since 33 mM pyruvate is gaining a more fundamental understanding of the is initially added and the solubility of oxygen in our processes involved in obtaining properly folded proteins reaction mixture is about 0.25 mM, significant oxygen in vitro. Another is continuing to understand and remove transfer is necessary for the production of acetyl phos- the limitations that lead to short reaction times in batch phate from pyruvate. This limitation is expected to be reactions. The third is the demonstration that cell-free worse at even larger scales because of the reduction in systems can be operated at increased production scales. surface-to-volume ratio. This work addresses producing proteins in vitro on a More recently, Kim and Swartz reported another novel milliliter, rather than microliter, scale in a batch system method for energy regeneration, the PANOx system (9). using two recently developed energy systems. The Yokoya- This system combines the conventional PEP system with ma group has recently shown the utility of continuous the addition of nicotinamide adenine dinucleotide (NAD) exchange cell-free methods in preparing milligram quan- and coenzyme A (CoA) to utilize both PEP and pyruvate tities of protein for crystal structure determination (5- to regenerate ATP (Figure 1B). The cofactors are neces- 7). However, production of milligram quantities of protein sary to exploit the endogenous enzymes present in the using a batch approach has not been pursued. E. coli extract for energy regeneration from pyruvate. The One of the schemes to regenerate energy is the pyru- PANOx system also increases the amino acid concentra- vate oxidase system (8). Kim and Swartz reported that tion as compared to the traditional PEP system (8) and pyruvate could be utilized to regenerate ATP during a supplements the reaction mixture with sodium oxalate, cell-free protein synthesis reaction. In this energy regen- which has been shown to enhance protein synthesis eration system, the pyruvate oxidase enzyme from either yields in batch reactions (10). Attempts to scale-up the Lactobacillus or Pediococcus sp. is added to the cell-free PANOx system have not been previously carried out, reaction mixture to convert pyruvate into acetyl-phos- although the challenges associated with the addition of phate. Molecular oxygen is required for this oxidation an exogenous enzyme and oxygen supply are not present. reaction. Acetyl-phosphate regenerates ATP via endog- In this work, production of 100 nmol quantities of active chloramphenicol acetyl transferase (CAT) is re- * To whom correspondence should be addressed. Tel: 650-723- ported with the use of both the pyruvate oxidase and the 5398. Fax: 650-725-0555. E-mail: [email protected]. PANOx energy regeneration system. To our knowledge, 10.1021/bp0341693 CCC: $27.50 © 2004 American Chemical Society and American Institute of Chemical Engineers Published on Web 11/20/2003 Biotechnol. Prog., 2004, Vol. 20, No. 1 103 Figure 1. Proposed mechanisms for secondary energy regeneration systems. (A) Pyruvate oxidase system. Pyruvate oxidase catalyzes the formation of acetyl phosphate from pyruvate, requiring molecular oxygen. Endogenous acetate kinase, present in the cell extract, transfers the phosphate group from acetyl phosphate to regenerate ATP. The phosphate generated during protein synthesis is recycled. (B) PANOx system. The conventional PEP system is coupled with ATP generation from pyruvate. This requires the addition of coenzymeA and NAD. Sodium oxalate is used to inhibit the energy wasting reaction that converts pyruvate to PEP. Abbreviations: acetate kinase (Ack), adenosine triphosphate (ATP), lactate dehydrogenase (Ldh), nicotinamide adenine dinucleotide (NAD), phosphoenolpyruvate (PEP), phosphoenolpyruvate synthase (Pps), pyruvate dehydrogenase (Pdh), phosphotransacetylase (Pta), pyruvate kinase (Pyk), pyruvate oxidase (Pox). this is the first account describing large scale cell-free Changes from the original A19 strain were carried out protein production using a fed-batch format. Rapid, by P1 phage transduction and include reverting a me- single-step affinity purification of CAT is also demon- thionine auxotrophy back to a methionine prototrophy, strated. These results begin to suggest the possibility for removing the speA gene encoding arginine decarboxylase large-scale production of protein pharmaceuticals using to stabilize arginine concentrations, removing the tnaA cell-free synthesis systems. In addition, these data sug- gene encoding trytophanase to stabilize tryptophan con- gest the feasibility of high-throughput batch protocols for centrations, and removing the tonA gene encoding the generating sufficient quantities and purity of proteins for ferrichrome-iron receptor to protect against phage infec- structure determination by NMR or X-ray cystallography. tion. When the cell-extract from this mutant A19 strain was used in a 3-h PANOx batch reaction, comparable Materials and Methods amounts of CAT were synthesized relative to reactions Cell-Free Protein Synthesis. Coupled transcription- when the cell extract was prepared from the original A19 translation reactions were carried out in 1.5 mL eppen- strain (data not shown). Extracts from cells grown on 2 dorf tubes or stirred glass beakers at 37 °C. The pyruvate x YTPG were shown to produce about 37% more protein oxidase reaction was performed in a 10 mL beaker, and and have reduced phosphatase activity relative to those the PANOx scale-up reactions were performed in a 25 from cells grown on2xYT(13). The standard reaction mL beaker. Plasmid pK7CAT was used as a template for mixture contains the following components: 57 mM protein synthesis. pK7CAT encodes for chloramphenicol Hepes-KOH (pH7.5), 1.2 mM ATP, 0.85 mM each of GTP, acetyl transferase (CAT), with the CAT gene cloned UTP and CTP, 1 mM dithiothreitol (DTT), 200 mM between the T7 promoter and the T7 terminator (9). The potassium glutamate, 80 mM ammonium acetate, 12 mM plasmid was purified using a QIAGEN Plasmid Maxi Kit magnesium acetate, 34 µg/mL folinic acid, 170.6 µg/mL (Valencia, CA). N-Terminal histidine-tagged T7 RNA E. coli tRNA mixture, 6.7 µg/mL plasmid, 33 µg/mL T7 polymerase was prepared from E. coli strain BL21 (using RNA polymerase, 11 µM L-[U-14C]-leucine, 2% poly- a modified form of the plasmid pAR1219 (11), containing (ethylene glycol) 8000, and 0.24 volume of S30 extract. the additional sequence for six N-terminal histidine The final total E. coli protein concentration in the residues) according to the procedures of Swartz et al. (12). reaction was 9.9 ( 0.7 mg/mL, as determined by a S30 extract for the pyruvate oxidase system was prepared Bradford assay using a commercially available assay from E. coli strain A19, a derivative of K12, as described reagent (Bio-Rad, Hercules, CA). Bovine serum albumin earlier (2). Extract for the PANOx system was prepared was used as the standard. In the pyruvate oxidase energy from E. coli strain A19 ∆tonA ∆speA ∆tnaA met+. Extract regeneration system, 33 mM sodium pyruvate, 0.5 mM preparation was carried out as above. However, cells were each of 20 unlabeled amino acids, 6 U/mL pyruvate grown on a glucose and phosphate media, 2 x YTPG (13). oxidase, 6.7 mM potassium phosphate, 3.3 mM thymidine 104 Biotechnol. Prog., 2004, Vol. 20, No. 1 triphosphate (TPP), and 0.3 mM flavin adenine dinucle- preparation for both methods was the same. Five percent otide (FAD) were also added. In the PANOx energy TCA at 4 °C was added to the cell extract reaction regeneration system, 33 mM phosphoenolpyruvate (PEP), mixture in a 1:1 volumetric ratio.