Scalable purification of Bacillus anthracis protective antigen from Escherichia coli

The anthrax toxin consists of three proteins, protective antigen (PA), lethal factor, and edema factor that are produced by the Gram-positive bacterium, Bacillus anthracis. Current vaccines against anthrax use PA as their primary component. In this study, we developed a scalable process to produce and purify multi-gram quantities of highly pure, recombinant PA (rPA) from Escherichia coli. The rPA protein was produced in a 50-L fermentor and purified to >99% purity using anion-exchange, hydrophobic interaction, and hydroxyapatite chromatography. The final yield of purified rPA from medium cell density fermentations resulted in approximately 2.7 g of rPA per kg of cell paste (approximately 270 mg/L) of highly pure, biologically active rPA protein. The results presented here exhibit the ability to generate multi-gram quantities of rPA from E. coli that may be used for the development of new anthrax vaccines and anthrax therapeutics.     

Production of biologically active Bacillus anthracis edema factor in Escherichia coli

Anthrax is caused by the gram-positive spore-forming bacterium Bacillus anthracis. The anthrax toxin consists of three proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF). PA facilitates the translocation of LF and EF into the cytosol of mammalian cells. LF is thought to be a zinc-dependent metalloprotease that results in death. EF is a calmodulin- and calcium-dependent adenylate cyclase that causes edema upon entrance into the cytosol by elevating the cAMP levels in cells. Previous efforts to produce recombinant EF (rEF) in Escherichia coli yielded only 2.5 mg of rEF per liter of culture. In this work, we produced soluble rEF in large quantities in both the periplasm and cytoplasm of E. coli from shake flasks and fermentors. The rEF protein was purified by standard chromatography and yielded >97% pure, biologically active rEF. Yields of purified rEF from medium cell density fermentations resulted in up to 2.38 g/L of highly pure, biologically active rEF protein. These results exhibit the ability to generate gram quantities of active rEF from E. coli.     

Design and preparation of non-tagged Yersinia pestis LcrV antigen in Escherichia coli and its immunogenicity in BALB/c mice

The whole encoding sequence for Yersinia pestis LcrV antigen was cloned into pET-32a(+) and expressed in Escherichia coli BL21 (DE3). The LcrV was high level expressed in the E. coli cytoplasm in a completely soluble form. Recombinant LcrV could be purified from the supernatant of the bacteria lysate after chromatography using a combination of Phenyl-Sepharose F F, DEAE-Sepharose F F and Hiload Superdex 75. The final yield of approximately 3 g of purified rLcrV from 42 L bioreactor containing 25 L LB medium was obtained. High-titer IgG directed against rLcrV was detected positive after immunization on the BALB/c mice. The results presented here exhibit the ability to generate multi-gram quantities of non-tagged rLcrV from E. coli that can be used for the development of vaccine for preventing plague.     

Recombinant murine growth hormone from E. coli inclusion bodies: Expression,high‐pressure solubilization and refolding,and characterization of activity and structure

We expressed recombinant murine growth hormone (rmGH) in E. coli as a cost‐effective way to produce large quantities (gram scale) of the protein for use in murine studies of immunogenicity to therapeutic proteins. High hydrostatic pressure was used to achieve high solubility and high refolding yields of rmGH protein produced in E. coli inclusion bodies. A two‐step column purification protocol was used to produce 99% pure monomeric rmGH. Secondary and tertiary structures of purified rmGH were investigated using circular dichroism and 2D‐UV spectroscopy. The purified rmGH produced was found to be biologically active in hypophysectomized rats. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Large-scale overproduction and rapid purification of the Escherichia coli ssb gene product. Expression of the ssb gene under lambda PL control

We report a rapid procedure for the large-scale purification of the Escherichia coli encoded single-strand binding (SSB) protein, helix-destabilizing protein which is essential for replication, recombination, and repair processes in E. coli. To facilitate the isolation of large quantities of the ssb gene product, we have subcloned the ssb gene into a temperature-inducible expression vector, pPLc28 [Remaut, E., Stanssens, P., & Fiers, W. (1981) Gene 15, 81-93], carrying the bacteriophage lambda PL promoter. A large overproduction of the ssb gene product results upon shifting the temperature of E. coli strains which carry the plasmid and also produce the thermolabile lambda cI857 repressor. After 5 h of induction, the ssb gene product represents approximately 10% of the total cell protein. The overexpression of the ssb gene and the purification protocol reported here enable one to isolate SSB protein (greater than 99% pure) with final yields of approximately 3 mg of SSB protein/g of cell paste. In fact, very pure (greater than 99%) SSB protein can be obtained after approximately 8 h, starting from frozen cells in the absence of any columns, although inclusion of a single-stranded DNA-cellulose column is generally recommended to ensure that the purified SSB protein possesses DNA binding activity. The ability to easily purify 1 g of SSB protein from 300-350 g of induced cells will facilitate physical studies requiring large quantities of this important protein.     

Optimization of inclusion body solubilization and renaturation of recombinant human growth hormone from Escherichia coli

Recombinant human growth hormone (r-hGH) was expressed in Escherichia coli as inclusion bodies. In 10 h of fed-batch fermentation, 1.6 g/L of r-hGH was produced at a cell concentration of 25 g dry cell weight/L. Inclusion bodies from the cells were isolated and purified to homogeneity. Various buffers with and without reducing agents were used to solubilize r-hGH from the inclusion bodies and the extent of solubility was compared with that of 8 M urea as well as 6 M Gdn-HCl. Hydrophobic interactions as well as ionic interactions were found to be the dominant forces responsible for the formation of r-hGH inclusion bodies during its high-level expression in E. coli. Complete solubilization of r-hGH inclusion bodies was observed in 100 mM Tris buffer at pH 12.5 containing 2 M urea. Solubilization of r-hGH inclusion bodies in the presence of low concentrations of urea helped in retaining the existing native-like secondary structures of r-hGH, thus improving the yield of bioactive protein during refolding. Solubilized r-hGH in Tris buffer containing 2 M urea was found to be less susceptible to aggregation during buffer exchange and thus was refolded by simple dilution. The r-hGH was purified by use of DEAE-Sepharose ion-exchange chromatography and the pure monomeric r-hGH was finally obtained by using size-exclusion chromatography. The overall yield of the purified monomeric r-hGH was approximately 50% of the initial inclusion body proteins and was found to be biologically active in promoting growth of rat Nb2 lymphoma cell lines.     

A combined approach to improving large-scale production of tobacco etch virus protease

Tobacco etch virus NIa proteinase (TEV protease) is an important tool for the removal of fusion tags from recombinant proteins. Production of TEV protease in Escherichia coli has been hampered by insolubility and addressed by many different strategies. However, the best previous results and newer approaches for protein expression have not been combined to test whether further improvements are possible. Here, we use a quantitative, high-throughput assay for TEV protease activity in cell lysates to evaluate the efficacy of combining several previous modifications with new expression hosts and induction methods. Small-scale screening, purification and mass spectral analysis showed that TEV protease with a C-terminal poly-Arg tag was proteolysed in the cell to remove four of the five arginine residues. The truncated form was active and soluble but in contrast, the tagged version was also active but considerably less soluble. An engineered TEV protease lacking the C-terminal residues 238-242 was then used for further expression optimization. From this work, expression of TEV protease at high levels and with high solubility was obtained by using auto-induction medium at 37 degrees C. In combination with the expression work, an automated two-step purification protocol was developed that yielded His-tagged TEV protease with >99% purity, high catalytic activity and purified yields of approximately 400 mg/L of expression culture (approximately 15 mg pure TEV protease per gram of E. coli cell paste). Methods for producing glutathione-S-transferase-tagged TEV with similar yields (approximately 12 mg pure protease fusion per gram of E. coli cell paste) are also reported.     

UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, functionally expressed in and purified from Escherichia coli, yeast, and insect cells

UDP-GlcNAc 2-epimerase/ManNAc kinase is the key enzyme of sialic acid biosynthesis in mammals. Its functional expression is a prerequisite for early embryogenesis and for the synthesis of several cell recognition motifs in adult organism. This bifunctional enzyme is involved in the development of different diseases like sialuria or hereditary inclusion body myopathy. For a detailed understanding of the enzyme, large amounts of the pure active protein are needed. Different heterologous cell systems were therefore analyzed for the enzyme, which was found to be functionally expressed in Escherichia coli, the yeast strains Saccharomyces cerevisiae and Pichia pastoris, and insect cells. In all these cell types, the expressed enzyme displayed both epimerase and kinase activities. In E. coli, up to 2mg protein/l cell culture was expressed, in yeast cells only 0.4mg/L, while up to 100mg/L, were detected in insect cells. In all three cell systems, insoluble protein aggregates were also observed. Purification from E. coli resulted in 100microg/L pure and structurally intact protein. For insect cells, purification methods were established which resulted in up to 50mg/L pure, soluble, and active protein. In summary, expression and purification of the UDP-GlcNAc 2-epimerase/ManNAc kinase in Sf-900 cells can yield the milligram amounts of protein required for structural characterization of the enzyme. However, the easier expression in E. coli and yeast provides sufficient quantities for enzymatic and kinetic characterization.     

Cloning and expression in Escherichia coli of the gene encoding Aspergillus flavus urate oxidase.

Amino acid sequencing of peptides obtained after proteolytic hydrolysis of Aspergillus flavus urate oxidase (uricase) permitted the design of oligodeoxynucleotide probes that were used to obtain 1.2- and 5-kilobase pair DNA fragments from A. flavus cDNA and genomic libraries, respectively. The cDNA fragment contained the entire coding region for uricase, and comparison with the genomic fragment revealed the presence of two short introns in the coding region of the gene. A. flavus uricase has around 40% overall identity with uricases from higher organisms but with many conserved amino acids. Hitherto highly conserved consensus patterns found in other uricases were found to be modified in the A. flavus enzyme, notably the sequence Val-Leu-Lys-Thr-Thr-Gln-Ser near position 150, which in the filamentous fungus is uniquely modified to Val-Leu-Lys-Ser-Thr-Asn-Ser. Silent mutations were introduced by cassette mutagenesis near the 5'-extremity of the coding sequence in order to conform with Escherichia coli codon usage, and the uricase was expressed in the E. coli cytoplasm in a completely soluble, biologically active form.     

Differential precipitation and zinc chelate chromatography purification of biologically active HTLV-I Tax1 expressed in E. coli

A protocol which involves sequential ammonium sulfate precipitation and zinc chelate chromatography to purify the HTLV-I Tax1 protein expressed in E. coli is described. The final Tax1 product is greater than 90% pure and the yield is approximately 1 mg per liter of liquid culture. The purified Tax1 protein is biologically active in indirect in vitro DNA binding assays and cellular NF-kB induction experiments.     

Expression, refolding, and purification of recombinant human phosphodiesterase 3B: definition of the N-terminus of the catalytic core

We have developed an expression, refolding, and purification protocol for the catalytic domain of human Phosphodiesterase 3B (PDE3B). High level expression in Escherichia coli has been achieved with yields of up to 20mg/L. The catalytic domain of the enzyme was purified by affinity chromatography utilizing a novel affinity ligand. PDE3B, purified by affinity chromatography, with no single impurity #10878;1% as determined by SDS-PAGE, has a specific activity of 2210+/-442nmol/min/mg and a KM for cAMP of 44+/-4.5nM. Reducing the size of the expressed catalytic domain from residues 387-1112 to residues 654-1086 greatly reduced the aggregation phenomena observed with the affinity purified PDE3B. The definition of the N-terminus of the catalytic core was examined through the generation of several truncation mutants spanning amino acid residues 636-674. Constructs starting at E665 and M674 were fully active and devoid of activity, respectively. A construct starting at D668 had a Vmax reduced by approximately 10-fold relative to the longer constructs, yet the KM was not affected. This indicates the minimal N-terminus of the catalytic core lies between E665 and Y667. Refolding and affinity purification of the 654-1073 catalytic core of PDE3B has been employed to produce large quantities of highly pure enzyme for structural studies.     

Expression in Escherichia coli of chemically synthesized gene for a novel opiate peptide alpha-neo-endorphin

Chemically synthesized alpha-neo-endorphin gene was fused to the Escherichia coli beta-galactosidase gene on the plasmid pKO13. The resulting recombinant DNA was used to transform E. coli cells. Radioimmunoassay for alpha-neo-endorphin in CNBr-treated bacterial cells showed that alpha-neo-endorphin was synthesized at approximately 5 x 10(5) molecules per single E. coli cell. One of the transformants, WA802/p alpha NE2, was used for alpha-neo-endorphin purification. From 10.9 g of wet cells, we isolated 4 mg of chemically pure and biologically active alpha-neo-endorphin.     

Refolding, purification, and characterization of a loop deletion mutant of human Bcl-2 from bacterial inclusion bodies

This report describes the cloning of recombinant human Bcl-2, in which the putative disordered loop region has been replaced with a flexible linker and the hydrophobic C-terminus has been replaced with a 6xHis tag (Bcl-2(6-32)-AAAA-Bcl-2(86-206)-HHHHHH, abbreviation rhBcl-2; amino acid numbering excludes the initiating methionine). This protein was expressed in Escherichia coli where it accumulated in insoluble form in inclusion bodies. After lysis the washed inclusion bodies were solubilized and an l-arginine assisted protein refolding route was employed to obtain biologically active protein. rhBcl-2 was purified further by nickel chelate chromatography to give protein of >95% purity, with an overall yield of 5 mg per g of E. coli cell paste. Edman sequencing showed that approximately 90% of the rhBcl-2 retained the initiating methionine residue. Analytical size exclusion chromatography suggested that the refolded and purified rhBcl-2 was monomeric in nondenaturing solution. Purified protein had an affinity for a Bax BH3 domain peptide comparable to that for in vivo folded recombinant human Bcl-2 and suppressed caspase activation in a cell-free assay for apoptosis. 1H NMR spectroscopy of rhBcl-2, both free and complexed with the Bax BH3 domain peptide, provided further evidence for the structural and functional integrity of the refolded protein. These findings parallel and extend those of Muchmore et al., who found that a loop deletion mutant of human Bcl-XL retained anti-apoptotic function.     

Trehalose-phosphate synthase of Mycobacterium tuberculosis. Cloning, expression and properties of the recombinant enzyme.

The trehalose-phosphate synthase (TPS) of Mycobacterium smegmatis was previously purified to apparent homogeneity and several peptides from the 58 kDa protein were sequenced. Based on that sequence information, the gene for TPS was identified in the Mycobacterium tuberculosis genome, and the gene was cloned and expressed in Escherichia coli with a (His)6 tag at the amino terminus. The TPS was expressed in good yield and as active enzyme, and was purified on a metal ion column to give a single band of approximately 58 kDa on SDS/PAGE. Approximately 1.3 mg of purified TPS were obtained from a 1-L culture of E. coli ( approximately 2.3 g cell paste). The purified recombinant enzyme showed a single band of approximately 58 kDa on SDS/PAGE, but a molecular mass of approximately 220 kDa by gel filtration, indicating that the active TPS is probably a tetrameric protein. Like the enzyme originally purified from M. smegmatis, the recombinant enzyme is an unusual glycosyltransferase as it can utilize any of the nucleoside diphosphate glucose derivatives as glucosyl donors, i.e. ADP-glucose, CDP-glucose, GDP-glucose, TDP-glucose and UDP-glucose, with ADP-glucose, GDP-glucose and UDP-glucose being the preferred substrates. These studies prove conclusively that the mycobacterial TPS is indeed responsible for catalyzing the synthesis of trehalose-P from any of the nucleoside diphosphate glucose derivatives. Although the original enzyme from M. smegmatis was greatly stimulated in its utilization of UDP-glucose by polyanions such as heparin, the recombinant enzyme was stimulated only modestly by heparin. The Km for UDP-glucose as the glucosyl donor was approximately 18 mm, and that for GDP-glucose was approximately 16 mm. The enzyme was specific for glucose-6-P as the glucosyl acceptor, and the Km for this substrate was approximately 7 mm when UDP-glucose was the glucosyl donor and approximately 4 mm with GDP-glucose. TPS did not show an absolute requirement for divalent cations, but activity was increased about twofold by 10 mm Mn2+. This recombinant system will be useful for obtaining sufficient amounts of protein for structural studies. TPS should be a valuable target site for chemotherapeutic intervention in tuberculosis.     

Highly purified recombinant varicella Zoster virus thymidine kinase is a homodimer

Recombinant varicella zoster virus (VZV) thymidine kinase (TK) was isolated in a fast and gentle two-step procedure from Escherichia coli. The TK was expressed as a PreScission-cleavable fusion protein and purified by glutathione and ATP affinity chromatography, yielding homogeneous, highly pure VZV TK. The purified enzyme displays enzymatic activities with K(m) values of 0.3 +/- 0.06 microM for the natural substrate thymidine and 11.6 +/- 3.2 microM for ATP, indicating the biochemical equivalence with the viral VZV TK expressed in infected cells. Determinations of the native molecular weight by size exclusion chromatography and native polyacrylamide gel electrophoresis revealed that the pure enzyme is biologically active as a homodimer.     

Cloning, sequence analysis and expression in Escherichia coli of the gene encoding a uricase from the yeast-like symbiont of the brown planthopper, Nilaparvata lugens

A urate oxidase (uricase; EC 1.7.3.3) gene of the yeast-like fungal endosymbiont of the brown planthopper, Nilaparvata lugens, was cloned, and sequenced together with its flanking regions. The gene comprised a open reading frame of 987 bp, that was split into two parts by a single 96 bp intron. The encoded uricase was 296 amino acids with 62% sequence identity with that of Aspergillus flavus. The molecular weight deduced was 32,882, and the predicted isoelectric point was 6.06. The symbiont's uricase conserved all the known consensus motifs, except the C-terminal PTS-1, Ser-basic-Leu. The leucine at the third position of PTS-1 was replaced by serine in the C-terminus of the symbiont's uricase. The symbiont's uricase gene was successfully expressed in Escherichia coli, and the product, tagged with histidine residues, was purified. The symbiont's uricase, thus produced, was as active as those from plants and animals, but less active than those from other fungi.     

Cloning and high-level expression of scorpion toxin BmKITa1 in Escherichia coli and insect cells

BmK ITa1 cDNA was cloned and highly expressed in E. coli and insect cell. SDS-PAGE and western blot analysis revealed that subunit molecular weight of expression products is about 40 kDa and 10 kDa respectively. The expression product purified by a Ni(2+)-IDA-sepharose 6B column was toxic for insect, which indicated that it was biologically activity. Furthermore, Quantitative estimation show that the biological activity of recombinant BmK ITa1 from Tn cells was more powerful than from E. coli.     

尿酸氧化酶基因的克隆、表达及其产物的应用

克隆了产朊假丝酵母(Candida utilis)AS2-117尿酸氧化酶(Urate Oxidase,Uricase,EC1.7.3.3)的基因。将此基因插入原核表达质粒pET21a后转化大肠杆菌BL21(DE3),获得高表达的重组转化子菌株。经IPTG诱导,重组尿酸酶基因表达量可达菌体可溶性蛋白的40%。重组尿酸氧化酶为有酶活性的可溶蛋白。Western印迹分析证实表达产物有免疫学活性。经DEAE DE52纤维素离子交换柱层析纯化,目的蛋白纯度可达95%。重组蛋白和天然蛋白的理化特征比较证明重组蛋白的热稳定性有较大提高。酶盒配制和临床应用实验表明重组蛋白可代替天然蛋白进行临床血清尿酸的分析。     

High-level expression and purification of biologically active recombinant pokeweed antiviral protein.

Pokeweed antiviral protein (PAP) from the leaves of the pokeweed plant, Phytolacca americana, is a naturally occurring single-chain ribosome-inactivating protein, which catalytically inactivates both prokaryotic and eukaryotic ribosomes. The therapeutic potential of PAP has gained considerable interest in recent years due to the clinical use of native PAP as the active moiety of immunoconjugates against cancer and AIDS. The clinical use of native PAP is limited due to inherent difficulties in obtaining sufficient quantities of a homogenously pure and active PAP preparation with minimal batch to batch variability from its natural source. Previous methods for expression of recombinant PAP in yeast, transgenic plants and Escherichia coli have resulted in either unacceptably low yields or were too toxic to the host system. Here, we report a successful strategy which allows high level expression of PAP as inclusion bodies in E. coli. Purification of refolded recombinant protein from solubilized inclusion bodies by size-exclusion chromatography yielded biologically active recombinant PAP (final yield: 10 to 12 mg/L). The ribosome depurinating in vitro N-glycosidase activity and cellular anti-HIV activity of recombinant PAP were comparable to those of the native PAP. This expression and purification system makes it possible to obtain sufficient quantities of biologically active and homogenous recombinant PAP sufficient to carry out advanced clinical trials. To our knowledge, this is the first large-scale expression and purification of biologically active recombinant PAP from E. coli.