Purification and biochemical characterization of the ErmSF macrolide-lincosamide-streptogramin B resistance factor protein expressed as a hexahistidine-tagged protein in Escherichia coli

The erm proteins confer resistance to the MLS (macrolide-lincosamide-streptogramin B) antibiotics in various microorganisms, including pathogens, through dimethylation of a single adenine residue (A2085: Bacillus subtilis coordinate) of the 23S rRNA to reduce the affinity of antibiotics, thereby enabling the cells to escape from the antibiotics' action, and this mechanism is predominantly adopted by microorganisms resistant to MLS antibiotics. ErmSF methyltransferase is one of the four gene products synthesized by Streptomyces fradiae NRRL 2338 to be resistant to its autogenous antibiotic, tylosin. In order to have a convenient source for the purification of milligram amounts, we expressed ErmSF in Escherichia coli using a T7 promoter-driven expression vector system, pET 23b, and the protein was expressed with a carboxy-terminal addition of six histidine residues in order to facilitate purification. Expression at 22 degrees C reduced the formation of insoluble aggregate, inclusion body, and resulted in accumulation of soluble hexahistidine-ErmSF up to 30% of total cell protein after 18 h. Metal-chelation chromatography yielded 126 mg of hexahistidine-ErmSF per liter of culture with a purity slightly greater than 95%. To examine the function of ErmSF in vivo and in vitro, its activity in E. coli (antibiotic susceptibility assay) andin vitro methyltransferase activity using in vitro-produced B. subtilis domain V, 434-, 257-, and 243-nt RNAs were investigated. The ErmSF in E. coli conferred resistance to erythromycin, whereas E. coli harboring an empty vector, pET23b, was susceptible. The purified recombinant protein successfully methylated domain V of 23S rRNA, which is known to contain all of the substrate elements recognized and to be methylated by erm proteins. However, the truncated substrates were methylated with decreased efficiencies. Almost all of domain V was monomethylated with less than 0.2 pM S-[methyl-(3)H]adenosylmethionine concentration. The roles of three structurally divided regions of domain V in recognition and methylation by ErmSF are proposed through kinetic studies using RNA substrates, in which each region is deleted, under the monomethylation condition.     

Purification and characterization of recombinant sTRAIL expressed in Escherichia coli

As a potential anti-tumor protein, tumor necrosis factor-related apoptosis-inducing ligand(TRAIL) has drawn considerable attention. This report presented the purification and characterization ofsoluble TRAIL, expressed as inclusion bodies in E. coli. sTRAIL inclusion bodies were solubilized andrefolded at a high concentration up to 0.9 g/L by a simple dilution method. Refolded protein was purifiedto electrophoretic homogeneity by a single-step immobilized metal affinity chromatography. The purifiedsTRAIL had a strong cytotoxic activity against human pancreatic tumor cell line 1990, with EDs0 about 1.5mg/L. Circular dichroism and fluorescence spectrum analysis showed that the refolded sTRAIL had astructure similar to that of native protein with 13-sheet secondary structure. This efficient procedure ofsTRAIL renaturation may be useful for the mass production of this therapeutically important protein.     

Purification and characterization of pea thioredoxin f expressed in Escherichia coli

The recently cloned cDNA for pea chloroplast thioredoxin f was used to produce, by PCR, a fragment coding for a protein lacking the transit peptide. This cDNA fragment was subcloned into a pET expression vector and used to transform E. coli cells. After induction with IPTG the transformed cells produce the protein, mainly in the soluble fraction of the broken cells. The recombinant thioredoxin f has been purified and used to raise antibodies and analysed for activity. The antibodies appear to be specific towards thioredoxin f and do not recognize other types of thioredoxin. The recombinant protein could activate two chloroplastic enzymes, namely NADP-dependent malate dehydrogenase (NADP-MDH) and fructose 1,6-bisphosphatase (FBPase), both using dithiothreitol as a chemical reductant and in a light-reconstituted/thylakoid assay. Recombinant pea thioredoxin f turned out to be an excellent catalyst for NADP-MDH activation, being the more efficient than a recombinant m-type thioredoxin of Chlamydomonas reinhardtii and the thioredoxin of E. coli. At the concentrations of thioredoxin used in the target enzyme activation assays only the recombinant thioredoxin f activated the FBPase.     

Purification and characterization of recombinant spider silk expressed in Escherichia coli

A partial cDNA clone, from the 3′ end of the dragline silk gene was isolated from Nephila clavipes major ampullate glands. This clone contains a 1.7-kb insert, consisting of a repetitive coding region of 1.4-kb and a 0.3-kb nonrepetitive coding region; 1.5-kb of the 1.7-kb fragment was cloned into Escherichia coli and a␣43-kDa recombinant silk protein was expressed. Characterization of the purified protein by Western blot, amino acid composition analysis, and matrix-assisted laser desorption ionization/time-of-flight mass spectrometry confirms it to be spider dragline silk. Received: 7 April 1997 / Received revision: 24 July 1997 / Accepted: 25 August 1997     

Purification and properties of Saccharomyces cerevisiae acetolactate synthase from recombinant Escherichia coli

The yeast ilv2 gene, encoding acetolactate synthase, was subcloned in an Escherichia coli expression vector. Although a major part of the acetolactate synthase synthesized by E. coli cells harbouring this vector was packaged into protein inclusion bodies, we used these recombinant E. coli cells to produce large quantities of the yeast enzyme. The yeast acetolactate synthase was purified to homogeneity using first streptomycin and ammonium sulfate precipitations, followed by T-gel thiophilic interaction, Sephacryl S-300 gel filtration, Mono Q anion exchange, and Superose 12 gel filtration chromatography. SDS/PAGE and gel filtration of the purified enzyme showed that it is a dimer composed of two subunits, each with the molecular mass of 75 kDa. The purified yeast acetolactate synthase was further characterized with respect to pH optimum, dependence of the substrate, pyruvate, and requirements of the cofactors, thiamin diphosphate, Mg2+, and FAD.     

Purification and characterization of human plasminogen activator inhibitor type I expressed in Saccharomyces cerevisiae

The rapidly acting inhibitor of plasminogen activators, PAI-1, was produced intracellularly in Saccharomyces cerevisiae by using the ADH2 promoter to drive the expression of the human PAI-1 cDNA. Approximately 8 mg of human PAI-1 was produced per liter of confluent yeast culture. A purification scheme which resulted in 20% recovery of isolated PAI-1 from the broken yeast cell homogenate was devised. Yeast-derived human PAI-1 differs from endothelial-type PAI-1 isolated from HT1080 fibrosarcoma cells in that the recombinant inhibitor does not contain carbohydrate side chains. Nevertheless, the activity and other functional attributes of yeast-derived PAI-1 are similar to those exhibited by HT1080 fibrosarcoma cell-derived PAI-1. Hence, this study demonstrates that expression of human PAI-1 in yeast is a viable strategy for the production of ample quantities of this key modulator of plasminogen activator-mediated proteolysis.     

Purification and characterization of a plant antimicrobial peptide expressed in Escherichia coli

MiAMP1 is a low-molecular-weight, cysteine-rich, antimicrobial peptide isolated from the nut kernel of Macadamia integrifolia. A DNA sequence encoding MiAMP1 with an additional ATG start codon was cloned into a modified pET vector under the control of the T7 RNA polymerase promoter. The pET vector was cotransformed together with the vector pSB161, which expresses a rare arginine tRNA. The peptide was readily isolated in high yield from the insoluble fraction of the Escherichia coli extract. The purified peptide was shown to have an identical molecular weight to the native peptide by mass spectroscopy indicating that the N-terminal methionine had been cleaved. Analysis by NMR spectroscopy indicated that the refolded recombinant peptide had a similar overall three-dimensional structure to that of the native peptide. The peptide inhibited the growth of phytopathogenic fungi in vitro in a similar manner to the native peptide. To our knowledge, MiAMP1 is the first antimicrobial peptide from plants to be functionally expressed in E. coli. This will permit a detailed structure-function analysis of the peptide and studies of its mode of action on phytopathogens.     

Purification and characterization of recombinant porcine prorelaxin expressed in Escherichia coli.

In this report we describe the purification and characterization of recombinant porcine prorelaxin expressed in Escherichia coli. Nucleotide sequence encoding porcine prorelaxin was inserted into an E. coli expression vector, pOTS, and the recombinant plasmid was transformed into the E. coli host (AR120). Upon induction with nalidixic acid, the 19-kDa recombinant porcine prorelaxin was produced at a level of approximately 8% of the total accumulated cell protein. The recombinant prorelaxin was purified to homogeneity by CM-cellulose chromatography and reversed-phase HPLC, after refolding in the presence of reduced and oxidized glutathione and a low concentration of guanidine-HCl. The identity of the recombinant prorelaxin was confirmed by the correct size, immunoreactivity with antibodies against native porcine relaxin, and direct amino-terminal sequence analysis. Furthermore, the purified recombinant prorelaxin could be converted to the 6-kDa relaxin by limited digestion with trypsin. Trypsin was shown to cleave at the carboxyl side of Arg29 and Arg137 residues of the recombinant prorelaxin, producing the des-ArgA1-B29-relaxin, and degrade the 13-kDa connecting peptide into small peptides. Both the recombinant prorelaxin and converted relaxin were found to be biologically active in an in vitro bioassay for relaxin.     

Purification and characterization of human H-ras proteins expressed in Escherichia coli.

The full-length normal and T24 mutant human H-ras proteins and two truncated derivatives of the T24 mutant were expressed efficiently in Escherichia coli. The proteins accumulated to 1 to 5% of total cellular protein, and each was specifically recognized by anti-ras monoclonal antibodies. The two full-length proteins as well as a carboxyl-terminal truncated derivative (deleted for 23 amino acid residues) were soluble upon cell lysis and were purified to 90% homogeneity without the use of denaturants. In contrast, an amino-terminal truncated ras derivative (deleted for 22 amino acid residues) required treatment with urea for its solubilization. The guanine nucleotide binding activity of these four proteins was assessed by a combination of ligand binding on proteins blots, immunoprecipitation, and standard filter binding procedures. The full-length proteins showed similar binding kinetics and a stoichiometry approaching 1 mol of GTP bound per mol of protein. The showed similar binding kinetics and a stoichiometry approaching 1 mol of GTP bound per mol of protein. The carboxyl-terminal truncated protein also bound GTP, but to a reduced extent, whereas the amino-terminal truncated protein did not have binding activity. Apparently, the carboxyl-terminal domain of ras, although important for transforming function, does not play a critical role in GTP binding.     

Purification and characterization of recombinant human interleukin-29 expressed in Escherichia coli

Human interleukin (IL)-29 is the latest member of the class II cytokine family. However, as a result of lacking efficient method to generate relatively large quantity of IL-29, little is known of its functions in man. In the present study, an Escherichia coli expression system for the rapid expression of the human IL-29 gene was developed. It involved of cloning IL-29 gene into the pET-44 Ek/LIC vector, which allowed expression of IL-29 with a fusion tag consisting of the NusA protein, polyhistidine and S peptide (Nus-His-S-tag), and introducing a thrombin recognition site between the fusion tag and IL-29. The expressed fusion protein was purified by S-protein agarose affinity chromatography, and the fusion tag was removed from recombinant IL-29 by cleavage with thrombin. The purified IL-29 appeared a single band on SDS-PAGE, and the yield of IL-29 was 60 mg from 1 l of bacterial culture. N-terminal sequencing confirmed the identity of the purified protein. The recombinant IL-29 showed specific antiviral activity that was comparable to the commercially available IFN alfa-2b preparation.     

Purification and characterization of the fusion protein trypsin-streptavidin expressed in Escherichia coli

Expression of fusion protein trypsin-streptavidin (TRYPSA)⁴ in Escherichia coli was evaluated and the protein purified. Protein expression was induced by 1 mM isopropylthio--D-galactoside (IPTG), and the enzyme activity was measured by the hydrolysis rate of p-toluenesulfonyl-l-arginine methyl ester (TAME). Expression of the fusion protein in the cell-free extract decreased with increased induction time; correspondingly, that in the inclusion bodies increased. The total expression in Luria–Bertani broth (LB) and Terrific Broth (TB) media reached the highest levels in 2 hr at 30°C. The optimum expression level was 35 and 48 U/L in LB and TB, respectively. Expression of the fusion protein was verified by Western Blot analysis using streptavidin antiserum, and the fusion protein was purified using a benzamidine Sepharose 6B affinity column at room temperature. The molecular size of the soluble purified fusion protein was determined by size-exclusion chromatography using Superose 12 FPLC. A molecular weight of 39–40 kDa was obtained, indicating that the soluble protein exists as a monomer; thus, the presence of the trypsin domain must prevent the streptavidin domain from tetramer formation.     

Purification and characterization of human uroporphyrinogen III synthase expressed in Escherichia coli

The side-chain asymmetry of physiological porphyrins is produced by the cooperative action of hydroxymethylbilane synthase and uroporphyrinogen (uro'gen) III synthase. Although the role of uro'gen III synthase is essential for the chemistry of porphyrin biosynthesis, many aspects, structural as well as mechanical, of uro'gen III synthase have yet to be studied. We report here an expression system in Escherichia coli and a purification procedure for human uro'gen III synthase. The enzyme in the lysate was unstable, but we found that glycerol prevents the activity loss in the lysate. The purified enzyme showed remarkable thermostability, particularly when kept in phosphate buffer containing DTT or EDTA, indicating that the enzyme activity may depend on its oxidation state. Examination of the relationship between the number of Cys residues that are accessible to 5,5'-dithiobis(2-nitrobenzoic acid) and the remaining activity during heat inactivation showed that a particular Cys residue is involved in activity loss. From the crystal structure of human uro'gen III synthase [Mathews et al. (2001) EMBO J. 20, 5832-5839], this Cys residue was considered to be Cys73, which is buried deep inside the enzyme, suggesting that Cys73 of human uro'gen III synthase plays an important role in enzyme activity.     

Purification and functional characterization of human 11beta hydroxylase expressed in Escherichia coli

The human 11beta-hydroxylase (hCYP11B1) is responsible for the conversion of 11-deoxycortisol into the major mammalian glucocorticoid, cortisol. The reduction equivalents needed for this reaction are provided via a short electron transfer chain consisting of a [2Fe-2S] ferredoxin and a FAD-containing reductase. On the biochemical and biophysical level, little is known about hCYP11B1 because it is very unstable for analyses performed in vitro. This instability is also the reason why it has not been possible to stably express it so far in Escherichia coli and subsequently purify it. In the present study, we report on the successful and reproducible purification of recombinant hCYP11B1 coexpressed with molecular chaperones GroES/GroEL in E. coli. The protein was highly purified to apparent homogeneity, as observed by SDS/PAGE. Upon mass spectrometry, the mass-to-charge ratio (m/z) of the protein was estimated to be 55 761, which is consistent with the value 55 760.76 calculated for the form lacking the translational initiator Met. The functionality of hCYP11B1 was analyzed using different methods (substrate conversion assays, stopped-flow, Biacore). The results clearly demonstrate that the enzyme is capable of hydroxylating its substrates at position 11-beta. Moreover, the determined NADPH coupling percentage for the hCYP11B1 catalyzed reactions using either 11-deoxycortisol or 11-deoxycorticosterone as substrates was approximately 75% in both cases. Biacore and stopped-flow measurements indicate that hCYP11B1 possesses more than one binding site for its redox partner adrenodoxin, possibly resulting in the formation of more than one productive complexes. In addition, we performed CD measurements to obtain information about the structure of hCYP11B1.     

Purification and characterization of recombinant Plasmodium falciparum adenylosuccinate synthetase expressed in Escherichia coli

Most parasitic protozoa lack the de novo purine biosynthetic pathway and rely exclusively on the salvage pathway for their purine nucleotide requirements. Enzymes of the salvage pathway are, therefore, candidate drug targets. We have cloned the Plasmodium falciparum adenylosuccinate synthetase gene. In the parasite, adenylosuccinate synthetase is involved in the synthesis of AMP from IMP formed during the salvage of the purine base, hypoxanthine. The gene was shown to code for a functionally active protein by functional complementation in a purA mutant strain of Escherichia coli, H1238. This paper reports the conditions for hyperexpression of the recombinant protein in E. coli BL21(DE3) and purification of the protein to homogeneity. The enzyme was found to require the presence of dithiothreitol during the entire course of the purification for activity. Glycerol and EDTA were found to stabilize enzyme activity during storage. The specific activity of the purified protein was 1143.6 +/- 36.8 mUnits/mg. The K(M)s for the three substrates, GTP, IMP, and aspartate, were found to be 4.8 microM, 22.8 microM, and 1.4 mM, respectively. The enzyme was a dimer on gel filtration in buffers of low ionic strength but equilibrated between a monomer and a dimer in buffers of increased ionic strength.     

Purification and characterization of human interleukin-1 beta expressed in recombinant Escherichia coli

The high-level expression of human interleukin-1 beta in Escherichia coli is described. The protein contributes about 12% of the total cell protein and is associated with the soluble cytoplasmic fraction of the cell. A method for the purification of the protein is given which is based on anion- and cation-exchange chromatographies. The isolated protein, shown to be homogeneous by several analytical methods, has been characterized by amino acid analysis, N- and C-terminal sequence analysis and analytical centrifugation. The protein is biologically active as demonstrated by two different in vitro assays, namely, the mononuclear cell factor (IL-1/MCF) assay and lymphocyte activating factor (IL-1/LAF) assay. The specific activities determined with the IL-1/MCF and IL-1/LAF assays, are 2 X 10(7) and 4 X 10(7) units mg-1, respectively.     

Purification and characterization of a Bacillus polymyxa beta-glucosidase expressed in Escherichia coli.

The beta-glucosidase encoded by the bglA gene from Bacillus polymyxa was overproduced in Escherichia coli by using a plasmid in which bglA is under control of the lacI promoter. Induction with isopropyl-beta-D-thiogalactopyranoside allowed an increase in the specific activity of the enzyme of about 100 times the basal level of gene expression. The enzyme was purified by a two-step procedure involving salting out with ammonium sulfate and ion-exchange chromatography with DEAE-cellulose. Fractions of beta-glucosidase activity recovered by this procedure, after electrophoresis in an acrylamide gel and staining with silver nitrate, yielded a single band of protein. This band was shown by a zymogram to correspond to beta-glucosidase activity. The purified protein showed an apparent molecular mass of 50 kDa and an isoelectric point of 4.6, values in agreement with those expected from the nucleotide sequence of the gene. Km values of the enzyme, with either cellobiose or p-nitrophenyl-beta-D-glucoside as the substrate, were determined. It was shown that the enzyme is competitively inhibited by glucose. The effects of different metallic ions and other agents were studied. Hg2+ was strongly inhibitory, while none of the other cations tested had any significant effect. Ethanol did not show the stimulating effect observed with other beta-glucosidases. The mechanism of enzyme action was investigated. High-pressure liquid chromatography analysis with cellobiose as the substrate confirmed previous data revealing the formation of two products, glucose and another, unidentified, compound. Results presented here indicate that this compound is cellotriose formed by transglycosylation.     

Production, purification, and characterization of human scFv antibodies expressed in Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli

Single chain (scFv) antibodies are used as affinity reagents for diagnostics, therapeutics, and proteomic analyses. The antibody discovery platform we use to identify novel antigen binders involves discovery, characterization, and production. The discovery and characterization components have previously been characterized but in order to fully utilize the capabilities of affinity reagents from our yeast surface display library, efforts were focused on developing a production component to obtain purified, soluble, and active scFvs. Instead of optimizing conditions to achieve maximum yield, efforts were focused on using a system that could quickly and easily produce and process hundreds of scFv antibodies. Heterologous protein expression in Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli were evaluated for their ability to rapidly, efficaciously, and consistently produce scFv antibodies for use in downstream proteomic applications. Following purification, the binding activity of several scFv antibodies were quantified using a novel Biacore assay. All three systems produced soluble scFv antibodies which ranged in activity from 0 to 99%. scFv antibody yields from Saccharomyces, Pichia, and E. coli were 1.5-4.2, 0.4-7.3, and 0.63-16.4 mgL(-1) culture, respectively. For our purposes, expression in E. coli proved to be the quickest and most consistent way to obtain and characterize purified scFv for downstream applications. The E. coli expression system was subsequently used to study three scFv variants engineered to determine structure-function relationships.     

Purification and characterization of recombinant Mortierella vinacea alpha-galactosidases I and II expressed in Saccharomyces cerevisiae.

The cDNAs coding for Mortierella vinacea alpha-galactosidases I and II were expressed in Saccharomyces cerevisiae under the control of the yeast GAL10 promoter. The recombinant enzymes purified to homogeneity from the culture filtrate were glycosylated, and had properties identical to those of the native enzymes except for improving the heat stability of alpha-galactosidase II and decreasing the specific activities of both enzymes.