Biomedical subjects

James R Swartz

Publications and source records attributed to James R Swartz.

At least 19 recordsLinked to original sources

PubMed2007

Cell-free synthesis of proteins that require disulfide bonds using glucose as an energy source.

The primary objective of this work was to create a cell-free protein synthesis extract that produces proteins requiring disulfide bonds while using glucose as an energy source. We attempted to avoid using iodoacetamide (IAM) to stabilize the required oxidizing thiol redox potential, since previous IAM pretreatments prevented glucose utilization apparently by inactivating glyceraldehyde 3-phosphate dehydrogenase (G-3PDH). Instead, the glutathione reductase (Gor)-mediated disulfide reductase system was disabled by deleting the gor gene from the KC6 cell-extract source strain. The thioredoxin reductase (TrxB)-mediated system was disabled by first adding a purification tag to the trxB gene in the chromosome to create strain KGK10 and then by affinity removal of the tagged TrxB. This was expected to result in a cell extract devoid of all disulfide reductase activity, but this was not the case. Although the concentration of IAM required to stabilize oxidized glutathione in the KGK10 extract could be reduced 20-fold, IAM pretreatment was still required to avoid disulfide reduction. Nonetheless, active urokinase and murine granulocyte macrophage-colony stimulating factor (mGM-CSF) were produced in reactions with KGK10 extract either with affinity removal of TrxB or with 50 microM IAM pretreatment. With the less intensive IAM pretreatment, glucose could be used as an energy source in a production system that promotes oxidative protein folding. This new protocol offers an economically feasible cell-free system for the production of secreted mammalian proteins as human therapeutics or vaccines.

Animals↗

PubMed2006

Cell-free production of scFv fusion proteins: an efficient approach for personalized lymphoma vaccines.

The unique immunoglobulin (Ig) idiotype on the surface of each B-cell lymphoma represents an ideal tumor-specific antigen for use as a therapeutic vaccine. We have used an Escherichia coli-based, cell-free protein-expression system to produce a vaccine within hours of cloning the Ig genes from a B-cell tumor. We demonstrated that a fusion protein consisting of an idiotypic single chain Fv antibody fragment (scFv) linked to a cytokine (GM-CSF) or to an immunostimulatory peptide was an effective lymphoma vaccine. These vaccines elicited humoral immune responses against the native Ig protein displayed on the surface of a tumor and protected mice against tumor challenge with efficacy equal to that of the conventional Ig produced in a mammalian cell and chemically coupled to keyhole limpet hemocyanin. The cell-free E coli system offers a platform for rapidly generating individualized vaccines, thereby allowing much more efficient application in the clinic.

Animals↗

PubMed2006

Simultaneous expression and maturation of the iron-sulfur protein ferredoxin in a cell-free system.

The model iron-sulfur (Fe-S) protein ferredoxin (Fd) from Synechocystis sp. PCC 6803 has been simultaneously produced and matured in a cell-free production system. After 6 h of incubation at 37 degrees C, Fd accumulated to >450 microg/mL. Essentially all was soluble, and 85% was active. Production and maturation of the protein in the cell-free system were found to be dependent in a coupled manner on the concentration of the supplemented iron and sulfur sources, ferrous ammonium sulfate and cysteine, respectively. The recombinant expression of ISC helper proteins during cell extract preparation did not increase cell-free Fd accumulation or activity, although the efficiency of iron and cysteine utilization increased. Fd maturation was independent of protein production rate, and proceeded at a constant rate throughout the period of active translation. In addition, incubation of denatured apo Fd with cell-free reaction components resulted in recovery of Fd activity, supporting the interpretation that maturation mechanisms did not act co-translationally. Incubation at 28 degrees C increased total and active protein accumulation, but decreased the ratio of active to total Fd produced. In summary, the high product yields and folding efficiency make the cell-free system described here an attractive platform for the study of Fe-S protein production and maturation. The system enables both small-volume, high throughput investigations as well as larger scale production. To our knowledge, this is the first demonstration of directed, high-yield production and maturation of an Fe-S protein in a cell-free system.

Cell-Free System↗

PubMed2006

Total amino acid stabilization during cell-free protein synthesis reactions.

Limitations in amino acid supply have been recognized as a substantial problem in cell-free protein synthesis reactions. Although enzymatic inhibitors and fed-batch techniques have been beneficial, the most robust way to stabilize amino acids is to remove the responsible enzymatic activities by genetically modifying the source strain used for cell extract preparation. Previous work showed this was possible for arginine, serine, and tryptophan, but cysteine degradation remained a major limitation in obtaining high protein synthesis yields. Through radiolabel techniques, we confirmed that cysteine degradation was caused by the activity of glutamate-cysteine ligase (gene gshA) in the cell extract. Next, we created Escherichia coli strain KC6 that combines a gshA deletion with previously described deletions for arginine, serine, and tryptophan stabilization. Strain KC6 grows well, and active cell extract can be produced from it for cell-free protein synthesis reactions. The extract from strain KC6 maintains stable amino acid concentrations of all 20 amino acids in a 3-h batch reaction. Yields for three different proteins improved 75-250% relative to cell-free expression using the control extract.

Amino Acids↗

PubMed2006

Rapid expression of functional genomic libraries.

Genomic-scale analysis of protein function is currently limited by the ability to rapidly express the enormous diversity of protein targets in their active form. We describe a method to construct transcriptionally active expression templates (ETs) in parallel using a single PCR step wherein the overlap-extension reaction for addition of transcription regulatory elements is separated from the amplification of the full-length product by using a GC-rich single primer. Over 90% of 55 diverse genomic targets were extended with T7 regulatory elements to form ETs in high yield and purity. The unpurified ETs directed protein expression using a cell-free protein synthesis (CFPS) system supplemented with cofactors and metal ions to activate a variety of enzymes. Higher activities were obtained in the modified CFPS reactions compared to standard reaction conditions. Protein purification was avoided because the expressed enzyme activity was significantly greater than the background activity associated with the cell extract. These improvements in the parallel synthesis of linear ETs combined with enhanced in vitro enzyme activation help to make CFPS systems more attractive platforms for high-throughput evaluation of protein function.

Bacteriophage T7↗

PubMed2005

Efficient and scalable method for scaling up cell free protein synthesis in batch mode.

A novel method for general cell free system scale-up in batch mode was applied to expression of E. coli chloramphenicol acetyl transferase (CAT) and a GMCSF-scFv fusion protein being developed as a B-cell lymphoma vaccine candidate (GLH). Performance of two different E. coli based cell-free systems was evaluated using the new scale-up approach. Reaction volumes from 15 to 500 microL were tested for both products and both reaction systems. In each case, the new scale-up method preserved total, soluble, and active volumetric yields of GLH and CAT at every reaction volume. At the 500 microL reaction volume, the PANOx SP system produced 560 +/- 36 microg/mL of active CAT and 99 +/- 10 microg/mL of active GLH protein using the new thin film approach whereas 500 microL test tube reactions produced 250 +/- 42 microg/mL and 72 +/- 7 microg/mL of active CAT and GLH respectively. Similarly, 500 microL cell-free synthesis reactions with the Cytomim system produced 481 +/- 38 microg/mL of active CAT and 109 +/- 15 microg/mL active GLH respectively in thin films compared to 29 +/- 7 microg/mL of active CAT and 5 +/- 2 microg/mL of active GLH protein in 500 microL test tube reactions. The new thin film approach improves oxygen supply for the Cytomim system, and increases the availability of hydrophobic surfaces. Analysis suggests that these surfaces provide significant benefit for protein expression and folding. We believe that this approach provides a general reaction scale-up technology that will be suitable for any protein target, cell free system, and reaction volume.

Animals↗

PubMed2005

Quantitative polysome analysis identifies limitations in bacterial cell-free protein synthesis.

Cell-free protein synthesis (CFPS) is becoming increasingly used for protein production as yields increase and costs decrease. CFPS optimization efforts have focused primarily on energy supply and small molecule metabolism, though little is known about the protein synthesis machinery or what limits protein synthesis rates. Here, quantitative polysome profile analysis was used to characterize cell-free translation, thereby elucidating many kinetic parameters. The ribosome concentration in Escherichia coli-based CFPS reactions was 1.6 +/- 0.1 microM, with 72 +/- 4% actively translating at maximal protein synthesis rate. A translation elongation rate of 1.5 +/- 0.2 amino acids per second per ribosome and an initiation rate of 8.2 x 10(-9) +/- 0.3 x 10(-9) M/s, which correlates to, on average, one initiation every 60 +/- 9 s per mRNA, were determined. The measured CFPS initiation and elongation rates are an order of magnitude lower than the in vivo rates and further analysis identified elongation as the major limitation. Adding purified elongation factors (EFs) to CFPS reactions increased the ribosome elongation rate and protein synthesis rates and yields, as well as the translation initiation rate, indicating a possible coupling between initiation and elongation. Further examination of translation initiation in the cell-free system showed that the first initiation on an mRNA is slower than subsequent initiations. Our results demonstrate that polysome analysis is a valid tool to characterize cell-free translation and to identify limiting steps, that dilution of translation factors is a limitation of CFPS, and that CFPS is a useful platform for making novel observations about translation.

Cell-Free System↗

PubMed2005

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.

Cell Culture Techniques↗

PubMed2005

Rapid expression of vaccine proteins for B-cell lymphoma in a cell-free system.

The idiotype (Id)-granulocyte-macrophage colony-stimulating factor (GM-CSF) fusion proteins are potential vaccines for immunotherapy of B-cell lymphoma. In this study, four vaccine candidates were constructed by fusing murine GM-CSF to the amino- or carboxy-terminus of the 38C13 murine B-lymphocyte Id scFv with two different arrangements of the variable regions of the heavy chain and light chain (VL-VH and VH-VL). scFv (VH-VL) and GM-CSF/scFv fusion proteins were expressed in an Escherichia coli cell-free protein synthesis system. In order to promote disulfide bond formation during cell-free expression, cell extract was pretreated with iodoacetamide (IAM), and a sulfhydryl redox buffer composed of oxidized and reduced glutathione was added. The E. coli periplasmic disulfide isomerase, DsbC, was also added to rearrange incorrectly formed disulfide linkages. The 38C13 B-lymphocyte Id scFv was expressed with 30% of its soluble yield in active form (43 microg/ml) when tested with an anti-idiotypic mAb, S1C5, as the capture antibody in radioimmunoassay. It was found that the amino-terminal GM-CSF fusion proteins, GM-VL-VH and GM-VH-VL, showed much higher activity than the carboxy-terminal GM-CSF fusion proteins, VL-VH-GM and VH-VL-GM, in stimulating the cell proliferation of a GM-CSF-dependent cell line, NFS-60. Between the two amino-terminal GM-CSF fusion proteins, GM-VL-VH showed a higher total and soluble yield than GM-VH-VL.

Animals↗

PubMed2004

Substrate replenishment extends protein synthesis with an in vitro translation system designed to mimic the cytoplasm.

Cytoplasmic mimicry has recently led to the development of a novel method for cell-free protein synthesis called the "Cytomim" system. In vitro translation with this new system produced more than a 5-fold yield increase of chloramphenicol acetyl transferase (CAT) relative to a conventional method using pyruvate as an energy substrate. Factors responsible for activating enhanced protein yields, and causes leading to protein synthesis termination have been assessed in this new system. Enhanced yields were caused by the combination of three changes: growing the extract source cells on 2x YTPG media versus 2x YT, replacing polyethylene glycol with spermidine and putrescine, and reducing the magnesium concentration from conventional levels. Cessation of protein synthesis was primarily caused by depletion of cysteine, serine, CTP, and UTP. Substrate replenishment of consumed amino acids, CTP, and UTP extended the duration of protein synthesis to 24 h in fed-batch operation and produced 1.2 mg/mL of CAT. By also adding more T7 RNA polymerase and plasmid DNA, yields were further improved to 1.4 mg/mL of CAT. These results underscore the critical role that nucleotides play in the combined transcription-translation reaction and highlight the importance of understanding metabolic processes influencing substrate depletion.

Biomimetics↗

PubMed2004

Enhancing multiple disulfide bonded protein folding in a cell-free system.

A recombinant plasminogen activator (PA) protein with nine disulfide bonds was expressed in our cell-free protein synthesis system. Due to the unstable and reducing environment in the initial E. coli-based cell-free system, disulfide bonds could not be formed efficiently. By treating the cell extract with iodoacetamide and utilizing a mixture of oxidized and reduced glutathione, a stabilized redox potential was optimized. Addition of DsbC, replacing polyethylene glycol with spermidine and putrescine to create a more natural environment, adding Skp, an E. coli periplasmic chaperone, and expressing PA at 30 degrees C increased the solubility of the protein product as well as the yield of active PA. Taken together, the modifications enabled the production of more than 60 microg/mL of bioactive PA in a simple 3-h batch reaction.

Cell-Free System↗

PubMed2004

Mimicking the Escherichia coli cytoplasmic environment activates long-lived and efficient cell-free protein synthesis.

Cell-free translation systems generally utilize high-energy phosphate compounds to regenerate the adenosine triphosphate (ATP) necessary to drive protein synthesis. This hampers the widespread use and practical implementation of this technology in a batch format due to expensive reagent costs; the accumulation of inhibitory byproducts, such as phosphate; and pH change. To address these problems, a cell-free protein synthesis system has been engineered that is capable of using pyruvate as an energy source to produce high yields of protein. The "Cytomim" system, synthesizes chloramphenicol acetyltransferase (CAT) for up to 6 h in a batch reaction to yield 700 microg/mL of protein. By more closely replicating the physiological conditions of the cytoplasm of Escherichia coli, the Cytomim system provides a stable energy supply for protein expression without phosphate accumulation, pH change, exogenous enzyme addition, or the need for expensive high-energy phosphate compounds.

Adenosine Triphosphate↗

PubMed2004

Cell-free production of active E. coli thioredoxin reductase and glutathione reductase.

Escherichia coli thioredoxin reductase (TR) and glutathione reductase (GR) are dimeric proteins that require a flavin adenine dinucleotide (FAD) cofactor for activity. A cell-free protein synthesis (CFPS) reaction supplemented with FAD was used to produce TR at 760 microg/ml with 89% of the protein being soluble. GR accumulated to 521 microg/ml in a cell-free reaction with 71% solubility. The TR produced was fully active with a specific activity of 1390 min(-1). The GR had a specific activity of 139 U/mg, which is significantly more active than reported for GR purified from cells. The specific activity for both TR and GR decreased without FAD supplementation. This research demonstrates that CFPS can be used to produce enzymes that are multimeric and require a cofactor.

Cell-Free System↗

PubMed2004

Efficient production of a bioactive, multiple disulfide-bonded protein using modified extracts of Escherichia coli.

In this report, we demonstrate that a complex mammalian protein containing multiple disulfide bonds is successfully expressed in an E.coli-based cell-free protein synthesis system. Initially, disulfide-reducing activities in the cell extract prevented the formation of disulfide bonds. However, a simple pretreatment of the cell extract with iodoacetamide abolished the reducing activity. This extract was still active for protein synthesis even under oxidizing conditions. The use of a glutathione redox buffer coupled with the DsbC disulfide isomerase and pH optimization produced 40 microg/mL of active urokinase protease in a simple batch reaction. This result not only demonstrates efficient production of complex proteins, it also emphasizes the control and flexibility offered by the cell-free approach.

Cell Extracts↗

PubMed2004

Genetic analysis of disulfide isomerization in Escherichia coli: expression of DsbC is modulated by RNase E-dependent mRNA processing.

We designed a selection strategy for the isolation of Escherichia coli mutants exhibiting enhanced protein disulfide isomerase activity. The folding of a variant of tissue plasminogen activator (v-tPA), a protein containing nine disulfide bonds, in the bacterial periplasm is completely dependent on the level of disulfide isomerase activity of the cell. Mutations that increase this activity mediate the formation of catalytically active v-tPA, which in turn cleaves a p-aminobenzoic acid (PABA)-peptide adduct to release free PABA and thus allows the growth of an auxotrophic strain. Following chemical mutagenesis, a total of eight E. coli mutants exhibiting significantly higher disulfide isomerization activity, not only with v-tPA but also with two other unrelated protein substrates, were isolated. This phenotype resulted from significantly increased expression of the bacterial disulfide isomerase DsbC. In seven of the eight mutants, the upregulation of DsbC was found to be related to defects in RNA processing by RNase E, the rne gene product. Specifically, the genetic lesions in five mutants were shown to be allelic to rne, while an additional two mutants exhibited impaired RNase E activity due to lesions in other loci. The importance of mRNA stability on the expression of DsbC is underscored by the short half-life of the dsbC transcript, which was found to be only 0.8 min at 37 degrees C in wild-type cells but was two- to threefold longer in some of the stronger mutants. These results (i) confirm the central role of DsbC in disulfide bond isomerization in the bacterial periplasm and (ii) suggest a critical role for RNase E in regulating DsbC expression.

Disulfides↗

PubMed2004

Cell-free protein synthesis with prokaryotic combined transcription-translation.

Cell-free biology exploits and studies complex biological processes in a controlled environment without intact cells. One model system is prokaryotic cell-free protein synthesis. This technology offers an attractive and convenient approach to produce properly folded recombinant DNA (rDNA) proteins on a laboratory scale, screen PCR fragment libraries in a high-throughput format, express pharmaceutical proteins, incorporate labeled or unnatural amino acids into proteins, and activate microbial physiology to allow for investigation of biological systems. We describe the preparation of materials necessary for the expression, quantification, and purification of rDNA proteins from active Escherichia coli extracts.

Autoradiography↗

PubMedDate not supplied

Rapid expression and purification of 100 nmol quantities of active protein using cell-free protein synthesis.

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 in a 5 mL reaction 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 chromatography 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.

Adenosine Triphosphate↗

PubMedDate not supplied

Affinity purification of lipid vesicles.

We present a novel column chromatography technique for recovery and purification of lipid vesicles, which can be extended to other macromolecular assemblies. This technique is based on reversible binding of biotinylated lipids to monomeric avidin. Unlike the very strong binding of biotin and biotin-functionalized molecules to streptavidin, the interaction between biotin-functionalized molecules and monomeric avidin can be disrupted effectively by ligand competition from free biotin. In this work, biotin-functionalized lipids (biotin-PEG-PE) were incorporated into synthetic lipid vesicles (DOPC), resulting in unilamellar biotinylated lipid vesicles. The vesicles were bound to immobilized monomeric avidin, washed extensively with buffer, and eluted with a buffer supplemented with free biotin. Increasing the biotinyl lipid molar ratio beyond 0.53% of all lipids did not increase the efficiency of vesicle recovery. A simple adsorption model suggests 1.1 x 10(13) active binding sites/mL of resin with an equilibrium binding constant of K = 1.0 x 10(8) M(-1). We also show that this method is very robust and reproducible and can accommodate vesicles of varying sizes with diverse contents. This method can be scaled up to larger columns and/or high throughput analysis, such as a 96-well plate format.

Adsorption↗