Arabidopsis Acetohydroxyacid Synthase Expressed in Escherichia coli Is Insensitive to the Feedback Inhibitors

Acetohydroxyacid synthase (AHAS), the first enzyme unique to the biosynthesis of isoleucine, leucine, and valine, is the target enzyme for several classes of herbicides. The AHAS gene from Arabidopsis thaliana, including the chloroplast transit peptide, was cloned into the bacterial expression plasmid pKK233-2. The resulting plasmid was used to transform an AHAS-deficient Escherichia coli strain MF2000. The growth of the MF2000 strain of E. coli was complemented by the functional expression of the Arabidopsis AHAS. The AHAS protein was processed to a molecular mass of 65 kilodaltons that was similar to the mature protein isolated from Arabidopsis seedlings. The AHAS activity extracted from the transformed E. coli cells was inhibited by imidazolinone and sulfonylurea herbicides. AHAS activity extracted from Arabidopsis is inhibited by valine and leucine; however, this activity was insensitive to these feedback inhibitors when extracted from the transformed E. coli.     

Expression of an Anaplerotic Enzyme, Pyruvate Carboxylase, Improves Recombinant Protein Production in Escherichia coli

Anaplerotic enzyme reactions are those which replenish tricarboxylic acid intermediates that are withdrawn for the synthesis of biomass. In this study, we examined recombinant protein production in Escherichia coli containing activity in an additional anaplerotic enzyme, pyruvate carboxylase. In batch fermentations, the presence of pyruvate carboxylase resulted in 68% greater production of the model protein, β-galactosidase, 41% greater cell yield, and 57% lower acetate concentration. We discuss why these results indicate that acetate concentration does not limit cell growth and protein synthesis, as predicted by other researchers, and suggest instead that the rate of acetate formation represents an inefficient consumption of glucose carbon, which is reduced by the presence of pyruvate carboxylase.     

The Histidyl-tRNA Synthetase from Streptococcus equisimilis: Overexpression in Escherichia coli,Purification, and Characterization

ABSTRACT

We describe the high-level expression of the Streptococcus equisimilis histidyl-tRNA synthetase gene hisS) in Escherichia coli and the purification and characterization of the gene product. Due to a lack of an efficient E. coli ribosome binding sequence in the hisS gene, the coding region was fused in-frame to the expression vector pT7-7, thereby creating a fusion gene construct (pT7-7recIII), which is under the control of a strong bacteriophage T7 promoter. Another construct (pT-7recII) was used for low level expression of the native histidyl-tRNA synthetase (HisRS). The plasmids were electroporated into E. coli HB101, which already contained pGP1-2. After temperature induction, the fusion HisRS, which has an extra 15 amino acids between the initiator Met and the second amino acid, Lys, was expressed at a level of —18% of total cell protein (～50 mg'liter of bacterial culture). The fusion HisRS was purified to >99% by a combination of anion exchange and cation exchange chromatography of the S100 fraction. The predicted MWs of the native and fusion proteins are 47,932 and 49,717, respectively. The mass of the active fusion HisRS was estimated to be 94,000 Da by Sephacryl S-200 gel filtration chromatography and 108,200 Da by nondenaturing PAGE. Both methods show that the funtional enzyme is a dimer of two identical subunits. SDS-PAGE analysis of purified fusion HisRS with or without reduction showed a single band of M_r = 53.7 kDa.     

Overexpression,Refolding, Purification of Erbin PDZ Domain from Escherichia coli and Preparation of its Polyclonal Antibody

Abstract

The Erbin was recently identified. The antibody against Erbin has not been commercially available. As a new member of peripheral protein LAP family and novel type of adaptor protein, its functions and binding partners are not completely known. In the present study, cDNA encoding PDZ domain of Erbin was inserted in a prokaryotic expression vector. His-tagged recombinant protein was overproduced in E. coli and purified by Ni-NTA column chromatography. About 14.4 mg of the purified protein was obtained from 500 mL of cell culture. The purity of the recombinant protein was higher than 90%. The polyclonal antibody against this protein was raised. The antibody can recognize both denatured and natural Erbin protein. It will be used to further identify the new binding partners of Erbin and study its unknown functions.     

Deoxycytidine Triphosphate Deaminase: Characterization of an Escherichia coli Mutant Deficient in the Enzyme

A mutant of Escherichia coli, previously shown to contain abnormal nucleoside triphosphate pools, was found to be defective in its ability to synthesize thymidine nucleotides. The defect is not in the enzyme thymidylate synthetase but in deoxycytidine triphosphate deaminase, an enzyme that supplies deoxyuridine monophosphate, the substrate for thymidylate synthetase.     

Sucrose-Phosphate Synthase from Synechocystis sp. Strain PCC 6803: Identification of the spsA Gene and Characterization of the Enzyme Expressed in Escherichia coli

The first identification and characterization of a prokaryotic gene (spsA) encoding sucrose-phosphate synthase (SPS) is reported for Synechocystis sp. strain PCC 6803, a unicellular non-nitrogen-fixing cyanobacterium. Comparisons of the deduced amino acid sequence and some relevant biochemical properties of the enzyme with those of plant SPSs revealed important differences in the N-terminal and UDP-glucose binding site regions, substrate specificities, molecular masses, subunit compositions, and regulatory properties.     

Cloning of ODAP Degrading Gene and Its Expression as Fusion Protein in Escherichia coli

A gene responsible for the degradation of ß-N-Oxalyl diaminopropionic acid (ODAP) was fused to the maIE gene, which codes for maltose binding protein, by cloning into an expression vector pMAL c2. The gene has been expressed as fusion protein of mol wt approximately 62 kD. It has been purified by affinity chromatography. The fusion protein has been cleaved by an endoprotease factor Xa and the presence of maltose binding protein and the product of the cloned gene confirmed. SDS-PAGE has shown that the product of the ODAP degrading gene is a single polypeptide of mol wt of about 20.7 kD.     

Triphenylmethane Reductase from Citrobacter sp. Strain KCTC 18061P: Purification, Characterization, Gene Cloning, and Overexpression of a Functional Protein in Escherichia coli

We purified to homogeneity an enzyme from Citrobacter sp. strain KCTC 18061P capable of decolorizing triphenylmethane dyes. The native form of the enzyme was identified as a homodimer with a subunit molecular mass of about 31 kDa. It catalyzes the NADH-dependent reduction of triphenylmethane dyes, with remarkable substrate specificity related to dye structure. Maximal enzyme activity occurred at pH 9.0 and 60°C. The enzymatic reaction product of the triphenylmethane dye crystal violet was identified as its leuco form by UV-visible spectral changes and thin-layer chromatography. A gene encoding this enzyme was isolated based on its N-terminal and internal amino acid sequences. The nucleotide sequence of the gene has a single open reading frame encoding 287 amino acids with a predicted molecular mass of 30,954 Da. Although the deduced amino acid sequence displays 99% identity to the hypothetical protein from Listeria monocytogenes strain 4b H7858, it shows no overall functional similarity to any known protein in the public databases. At the N terminus, the amino acid sequence has high homology to sequences of NAD(P)H-dependent enzymes containing the dinucleotide-binding motif GXXGXXG. The enzyme was heterologously expressed in Escherichia coli, and the purified recombinant enzyme showed characteristics similar to those of the native enzyme. This is the first report of a triphenylmethane reductase characterized from any organism.     

Characterization of ChpBK, an mRNA interferase from Escherichia coli

Escherichia coli contains a number of antitoxin-toxin modules on its chromosome, which are responsible for cell growth arrest and possible cell death. ChpBK is a toxin encoded by the ChpBIK antitoxin-toxin module. This module consists of a pair of genes, chpBI and chpBK encoding antitoxin ChpBI and toxin ChpBK, respectively. ChpBK consists of 116 amino acid residues, and its sequence shows 35% identity and 52% similarity to MazF, another E. coli toxin. MazF has been shown to be a sequence-specific (ACA) endoribonuclease that cleaves cellular mRNAs and effectively blocks protein synthesis and is thus termed as an mRNA interferase. Here we demonstrate that ChpBK is another mRNA interferase in E. coli whose induction effectively blocks cell growth in a manner similar to that of MazF. The protein synthesis as judged by incorporation of [35S]methionine was, however, reduced by only 60% upon ChpBK induction. We demonstrate that ChpBK is a new sequence-specific endoribonuclease that cleaves mRNAs both in vivo and in vitro at the 5'-or3'-side of the A residue in ACY sequences (Y is U, A, or G). The ChpBK cleavage of a synthetic RNA substrate generated a 2',3'-cyclic phosphate group at the 3'-end of the 5'-end product and a 5'-OH group at the 5'-end of the 3'-end product in a manner identical to that of MazF.     

Characterization of CpdC,a Large-Ring Lactone-Hydrolyzing Enzyme from Pseudomonas sp. Strain HI-70, and Its Use as a Fusion Tag Facilitating Overproduction of Proteins in Escherichia coli

There are few entries of carbon-carbon bond hydrolases (EC 3.7.1.-) in the ExPASy database. In microbes, these enzymes play an essential role in the metabolism of alicyclic or aromatic compounds as part of the global carbon cycle. CpdC is a ω-pentadecalactone hydrolase derived from the degradation pathway of cyclopentadecanol or cyclopentadecanone by Pseudomonas sp. strain HI-70. CpdC was purified to homogeneity and characterized. It is active as a dimer of 56,000 Da with a subunit molecular mass of 33,349. Although CpdC has the highest activity and reaction rate (k_cat) toward ω-pentadecalactone, its catalytic efficiency favors lauryl lactone as a substrate. The melting temperature (T_m) of CpdC was estimated to be 50.9 ± 0.1°C. The half-life of CpdC at 35°C is several days. By virtue of its high level of expression in Escherichia coli, the intact CpdC-encoding gene and progressive 3′-end deletions were employed in the construction of a series of fusion plasmid system. Although we found them in inclusion bodies, proof-of-concept of overproduction of three microbial cutinases of which the genes were otherwise expressed poorly or not at all in E. coli was demonstrated. On the other hand, two antigenic proteins, azurin and MPT63, were readily produced in soluble form.     

Cloning and Functional Expression of a Novel Geranylgeranyl Pyrophosphate Synthase Gene from Arabidopsis thaliana in Escherichia coli

A gene encoding a novel geranylgeranyl pyrophosphate (GGPP)synthase from Arabidopsis thaliana has been identified and termedGGPS5. The gene has been sequenced and expressed in Escherichiacoli. The deduced amino acid sequence showed 64.5% and 57.5%identity with a putative GGPP synthase from Arabidopsis andCapsicum annuum, respectively. GGPP enzymatic activity was detectedin E. coli cells expressing the GGPS5 gene in two differentways. One was the direct measurement of GGPP synthase activityin cell extracts and the other was the yellow color productionof cells when the GGPS5 gene was co-expressed with crtB, crtI,crtX, crtY and crtZ genes derived from Erwinia uredovora. (Received May 20, 1996; Accepted December 14, 1996)     

Characterization of an Extremely Thermostable Restriction Enzyme, PspGI, from a Pyrococcus Strain and Cloning of the PspGI Restriction-Modification System in Escherichia coli

An extremely thermostable restriction endonuclease, PspGI, was purified from Pyrococcus sp. strain GI-H. PspGI is an isoschizomer of EcoRII and cleaves DNA before the first C in the sequence 5′ ^CCWGG 3′ (W is A or T). PspGI digestion can be carried out at 65 to 85°C. To express PspGI at high levels, the PspGI restriction-modification genes (pspGIR and pspGIM) were cloned in Escherichia coli. M.PspGI contains the conserved sequence motifs of α-aminomethyltransferases; therefore, it must be an N4-cytosine methylase. M.PspGI shows 53% similarity to (44% identity with) its isoschizomer, M.MvaI from Micrococcus variabilis. In a segment of 87 amino acid residues, PspGI shows significant sequence similarity to EcoRII and to regions of SsoII and StyD4I which have a closely related recognition sequence (5′ ^CCNGG 3′). PspGI was expressed in E. coli via a T7 expression system. Recombinant PspGI was purified to near homogeneity and had a half-life of 2 h at 95°C. PspGI remained active following 30 cycles of thermocycling; thus, it can be used in DNA-based diagnostic applications.     

Purification and Characterization of the Crown Gall-specific Enzyme, Octopine Synthase

A single enzyme catalyzes the synthesis of all four N²-(1-carboxyethyl)-amino acid derivatives found in a crown gall tumor tissue induced by Agrobacterium tumefaciens (E. F. Sm. and Town.) Conn strain B6 on sunflower (Helianthus annuus L.). This enzyme, octopine synthase, has been purified by ammonium sulfate fractionation and chromatography on diethylaminoethylcellulose, blue agarose, and hydroxylapatite. The purified enzyme has all the N²-(1-carboxyethyl)-amino acid synthesizing activities found in crude preparations, and the relative activities with six amino acids remain nearly constant during purification. Although the maximum velocities (V) and Michaelis constants (K_m) differ, the ratio V/K_m is the same for all amino acid substrates. Thus an equimolar mixture of amino acids will give rise to an equimolar mixture of products. The kinetic properties of the enzyme are consistent with a partially ordered mechanism with arginine (NADPH, then arginine or pyruvate). Octopine synthase is a monomeric enzyme with a molecular weight of 39,000 by gel filtration and 38,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Preparation and Characterization of Recombinant Murine p65/L-Plastin Expressed in Escherichia coli and High-titer Antibodies against the Protein

We previously identified a 65-kDa protein (p65) that was phosphorylated in activated macrophages. It has turned out to be a murine homologue of human L-plastin, which was identified as a novel protein in human cancer cells. p65/L-plastin is characterized by a series of Ca²⁺-, calmodulin-, and actin-binding domains, and is thought to play a crucial role in leukocytes and cancer cells. We have expressed a recombinant (r) p65/L-plastin in Escherichia coli that binds to β-actin and prepared high-titer antibodies using large amounts of the protein as immunogen. Anti-rp65/L-plastin antibodies recognize native p65/L-plastin as well as rp65/L-plastin and have enabled us to detect the fine structures of intracellular p65/L-plastin, and it was found that its localization was extensively changed by stimulation with bacterial components. We further developed an enzyme-linked immunosorbent assay system and a flow cytometry method using these reagents, which made it possible to measure antibodies, including autoantibodies, against p65/L-plastin and to evaluate the maturation-dependent expression of the protein in leukocytes.     

Purification, Characterization, and Immunological Properties of NADH-Dependent Glutamate Synthase from Rice Cell Cultures

To obtain a monospecific antibody against NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14), the enzyme was purified to homogeneity from cultured rice cells (Oryza sativa) with column chromatography using Butyl Toyopearl 650M, Sephacryl S-300, Blue Sepharose CL-6B, and Butyl Toyopearl 650S. The specific activity at the final stage of the purification was 9.8 micromoles of glutamate formed per minute per milligram of protein. The yield was 6.1% and purification was 815-fold. Analysis by denaturing gel electrophoresis revealed a single polypeptide with an apparent molecular weight of 196,000, similar to the value of 194,000 estimated for the native protein. Apparent K_m values for l-glutamine, 2-oxoglutarate, and NADH were 811, 76, and 3.0 micromolar, respectively. Neither NADPH nor l-asparagine substituted for NADH and l-glutamine, respectively. The enzyme had its absorption maxima at 273, 373, and 440 nanometers with a shoulder at 475 nanometers, suggesting that the rice NADH-GOGAT is a flavoprotein. Monospecific antibody raised against NADH-GOGAT purified from the rice cells was obtained as the first instance for the enzyme in higher plants. Immunological analyses showed that the antibody for rice cell NADH-GOGAT reacted with only the enzyme in extracts from the cells. The anti-NADH-GOGAT antibody did not recognize the ferredoxin-GOGAT purified from rice leaves, and likewise the anti-rice leaf ferredoxin-GOGAT antibody did not react with the NADH-GOGAT purified from the cultured rice cells.     

Overexpression and Purification of the Escherichia coli Inner Membrane Enzyme Acyl–Acyl Carrier Protein Synthase in an Active Form

Acyl–acyl carrier protein synthase (Aas) is widely used to synthesize thioester adducts of fatty acids between 8 and 18 carbons in length enzymatically to the phosphopantetheine group of acyl carrier protein. The enzyme is an 80.6-kDa inner membrane protein that functions in vivo as a 2-acylglycerophosphoethanolamine acyltransferase. The E. coli aas open reading frame was inserted into the expression plasmid pET28a so that, upon expression, a 21-amino-acid extension containing 6 consecutive histidine residues was added to the carboxyl terminus. The plasmid was designated pAasH. The activity of Aas in membranes was assessed from several cell lines. Membranes from the commonly used host line BL21(DE3) containing pAasH accumulated 30-fold and 38-fold more Aas activity than membranes from BL21(DE3) cells lacking the plasmid, when induced with isopropyl β- -thiogalactopyranoside (IPTG) or lactose, respectively. When pAasH was expressed under IPTG induction in cell line C41(DE3), a previously described cell line selected to enhance the expression of membrane proteins, Aas levels accumulated to 135-fold higher levels than in the cell line lacking the plasmid. Functional Aas can be isolated from either BL21(DE3) or C41(DE3) cell lines by differential centrifugation, followed by detergent extraction with Triton X-100 and nickel nitrilotriacetic acid affinity chromatography. The overexpression of Aas in cell line C41(DE3) is noteworthy compared to cell line BL21(DE3) because it results in a 3- to 4-fold higher accumulation of active enzyme in the membrane fraction and a lower proportion of inactive protein in the inclusion body.     

Overexpression, purification, and crystallization of the membrane-bound fumarate reductase from Escherichia coli

Quinol-fumarate reductase (QFR) from Escherichia coli is a membrane-bound four-subunit respiratory protein that shares many physical and catalytic properties with succinate-quinone oxidoreductase (EC 1.3.99.1) commonly referred to as Complex II. The E. coli QFR has been overexpressed using plasmid vectors so that more than 50% of the cytoplasmic membrane fraction is composed of the four-subunit enzyme complex. The growth characteristics required for optimal levels of expression with minimal degradation by host cell proteases and oxidation factors were determined for the strains harboring the recombinant plasmid. The enzyme is extracted from the enriched membrane fraction using the nonionic detergent Thesit (polyoxyethylene(9)dodecyl ether) in a monodisperse form and then purified by a combination of anion-exchange, perfusion, and gel filtration chromatography. The purified enzyme is highly active and contains all types of redox cofactors expected to be associated with the enzyme. Crystallization screening of the purified QFR by vapor diffusion resulted in the formation of crystals within 24 h using a sodium citrate buffer and polyethylene glycol precipitant. The crystals contain the complete four-subunit QFR complex, diffract to 3.3 A resolution, and were found to be in space group P2(1)2(1)2(1) with unit cell dimensions a = 96.6 A, b = 138.1 A, and c = 275.3 A. The purification and crystallization procedures are highly reproducible and the general procedure may prove useful for Complex IIs from other sources.     

Strategies for the Production of Recombinant Protein in Escherichia coli

In the recent past years, a large number of proteins have been expressed in Escherichia coli with high productivity due to rapid development of genetic engineering technologies. There are many hosts used for the production of recombinant protein but the preferred choice is E. coli due to its easier culture, short life cycle, well-known genetics, and easy genetic manipulation. We often face a problem in the expression of foreign genes in E. coli. Soluble recombinant protein is a prerequisite for structural, functional and biochemical studies of a protein. Researchers often face problems producing soluble recombinant proteins for over-expression, mainly the expression and solubility of heterologous proteins. There is no universal strategy to solve these problems but there are a few methods that can improve the level of expression, non-expression, or less expression of the gene of interest in E. coli. This review addresses these issues properly. Five levels of strategies can be used to increase the expression and solubility of over-expressed protein; (1) changing the vector, (2) changing the host, (3) changing the culture parameters of the recombinant host strain, (4) co-expression of other genes and (5) changing the gene sequences, which may help increase expression and the proper folding of desired protein. Here we present the resources available for the expression of a gene in E. coli to get a substantial amount of good quality recombinant protein. The resources include different strains of E. coli, different E. coli expression vectors, different physical and chemical agents and the co expression of chaperone interacting proteins. Perhaps it would be the solutions to such problems that will finally lead to the maturity of the application of recombinant proteins. The proposed solutions to such problems will finally lead to the maturity of the application of recombinant proteins.     

Overexpression, purification, and characterization of UDP-N-acetylmuramyl:L-alanine ligase from Escherichia coli.

UDP-N-acetylmuramyl:L-alanine ligase from Escherichia coli was overexpressed more than 600-fold and purified to near homogeneity. The purified enzyme was found to ligate L-alanine, L-serine, and glycine, as well as the nonnatural amino acid beta-chloro-L-alanine, to UDP-N-acetylmuramic acid. On the basis of (i) the specificity constants of the enzyme determined for L-alanine, L-serine, and glycine and (ii) the levels of these amino acids in the intracellular pool, it was calculated that the rates of incorporation of L-serine and glycine into peptidoglycan precursor metabolites could maximally amount to 0.1 and 0.5%, respectively, of the rate of L-alanine incorporation.     

Immunological Characterization of Honey Proteins and Identification of MRJP 1 as an IgE-Binding Protein

We encountered a fourth case of honey allergy in Japan. We characterized and identified the IgE-binding proteins in honey using the serum of a honey-allergenic patient. Immunoblot analysis revealed that IgE in the patient serum specifically bound to four proteins in each honey sample. At least three of these IgE-binding proteins were N-linked glycoproteins. To identify the 60-kDa IgE-binding protein in dandelion honey, the N-terminal sequences of the fragmented protein were analyzed, revealing the protein to be major royal jelly protein 1 (MRJP 1). Three IgE-binding proteins removed of N-linked oligosaccharide showed a large reduction in IgE-binding activity as compared with the intact protein. This suggests that the carbohydrates in the IgE-binding proteins are a major epitope for patient IgE.