Hydantoin racemase from Arthrobacter aurescens DSM 3747: heterologous expression, purification and characterization

In Arthrobacter aurescens DSM 3747 three enzymes are involved in the complete conversion of slowly racemizing 5'-monosubstituted D,L-hydantoins to L-amino acids, a stereoselective hydantoinase, a stereospecific L-N-carbamoylase and a hydantoin racemase. The gene encoding the hydantoin racemase, designated hyuA, was identified upstream of the previously described L-N-carbamoylase gene in the plasmid pAW16 containing genomic DNA of A. aurescens. The gene hyuA which encodes a polypeptide of 25.1 kDa, was expressed in Escherichia coli and the recombinant protein purified to homogeneity and further characterized. The optimal condition for racemase activity were pH 8.5 and 55 degrees C with L-5-benzylhydantoin as substrate. The enzyme was completely inhibited by HgCL2 and iodoacetamide and stimulated by addition of dithiothreitol. No effect on enzyme activity was seen with EDTA. The enzyme showed preference for hydantoins with arylalkyl side chains. Kinetic studies revealed substrate inhibition towards the aliphatic substrate L-5-methylthioethylhydantoin. Enzymatic racemization of D-5-indolylmethylenehydantoin in D2O and NMR analysis showed that the hydrogen at the chiral center of the hydantoin is exchanged against solvent deuterium during the racemization.     

Immobilization of the hydantoin cleaving enzymes from Arthrobacter aurescens DSM 3747.

The immobilization procedure of the two industrially important hydantoin cleaving enzymes--hydantoinase and L-N-carbamoylase from Arthrobacter aurescens DSM 3747--was optimized. Using different methods (carbodiimide, epoxy activated carriers) it was possible to immobilize the crude hydantoinase from A. aurescens DSM 3747 to supports containing primary amino groups with a yield of up to 60%. Immobilization on more hydrophobic supports such as Eupergit C and C 250 L resulted in lower yields of activity, whereas the total protein coupled remained constant. All attempts to immobilize the crude L-N-carbamoylase resulted in only low activity yields. Therefore, the enzyme was highly purified and used in immobilization experiments. The pure enzyme could easily be obtained in large amounts by cultivation of a recombinant Escherichia coli strain following a three step purification protocol consisting of cell disruption, chromatography on Streamline diethylaminoethyl and Mono Q. The immobilization of the L-N-carbamoylase was optimized with respect to the coupling yield by varying the coupling method as well as the concentrations of protein, carrier and carbodiimide. Using 60 mM of water-soluble carbodiimide, nearly 100% of the enzyme activity and protein could be immobilized to EAH Sepharose 4B.     

Cloning, nucleotide sequence and expression of a hydantoinase and carbamoylase gene from Arthrobacter aurescens DSM 3745 in Escherichia coli and comparison with the corresponding genes from Arthrobacter aurescens DSM 3747

The genes encoding hydantoinases (hyuH1) and carbamoylases (hyuC1) from Arthrobacter aurescens DSM 3745 and Arthrobacter aurescens DSM 3747 (hyuH2, hyuC2) were cloned in Escherichia coli and the nucleotide sequences determined. The hydantoinase genes comprised 1,377 base pairs and the carbamoylase genes 1,239 base pairs each. Both hydantoinases, as well as both carbamoylases, showed a high degree of nucleotide and amino acid sequence identity (96-98%). The hyuH and hyuC genes were expressed in E. coli under the control of the rhamnose promoter and the different specific activities obtained in E. coli crude extracts were compared to those produced by the original hosts. For purification the hyuH2 gene was expressed as a maltose-binding protein (MalE) and as an intein-chitin binding domain (CBD) fusion in E. coli. The expression of malE-hyuH2 resulted in the production of more soluble and active protein. With respect to temperature stability, optimal pH and optimal temperature, substrate and stereospecificity, the purified fusion enzyme exhibited properties similar to those of the wild-type enzyme.     

Cloning, nucleotide sequence and expression of a new L-N-carbamoylase gene from Arthrobacter aurescens DSM 3747 in E. coli

An L-N-carbamoyl amino acid amidohydrolase (L-N-carbamoylase) from Arthrobacter aurescens DSM 3747 was cloned in E. coli and the nucleotide sequence was determined. After expression of the gene in E. coli the enzyme was purified to homogeneity and characterized. The enzyme was shown to be strictly L-specific and exhibited the highest activity in the hydrolysis of beta-aryl substituted N alpha-carbamoyl-alanines as e.g. N-carbamoyl-tryptophan. Carbamoyl derivatives of beta-alanine and charged aliphatic amino acids were not accepted as substrates. The N-carbamoylase of A. aurescens DSM 3747 differs from all known enzymes with respect to its substrate specificity although amino acid sequence identity scores of 35-38% to other N-carbamoylases have been detected. The enzyme consists of two subunits of 44,000 Da, and has an isoelectric point of 4.3. The optima of temperature and pH were determined to be 50 degrees C and pH 8.5 respectively. At 37 degrees C the enzyme was completely stable for several days.     

Catalytic and structural function of zinc for the hydantoinase from Arthrobacter aurescens DSM 3745

Metal dependency of the hydantoin amidohydrolase (hydantoinase) from Arthrobacter aurescens DSM 3745 has been analyzed based on kinetic studies of metal/chelator-caused enzyme inactivation, denaturation and reactivation, accompanied by the identification of specific metal binding ligands. The enzyme can be inactivated by metal chelating agents and—apart from the loss of its activity—completely dissociates into its subunits. Enzyme activity can be restored from recollected monomers by the addition of cobalt, manganese or zinc-ions, whereas nickel and magnesia remain ineffective. Subjection of the hydantoinase to metal analysis reveals a content of 10 mol zinc per mol enzyme. Zinc plays an essential role not only for the catalytic activity but also for the stabilization of the active quarternary structure of the hydantoinase. Histidine-specific chemical modification of the enzyme causes a complete loss of the catalytic activity and reveals histidine residues as putative zinc binding ligands. Both, the metal/chelator-caused enzyme inactivation as well as the metal-caused enzyme reactivation, can be reduced in the presence of the substrate. Therefore, it is very likely that at least one metal-ion acts specifically near or at the active site of the enzyme.     

Amino acid composition and physiochemical characterization of chondroitinase from Arthrobacter aurescens.

The amino acid and carbohydrate compositions of chondroitinase AC [EC 4.2.2.5] from Arthrobacter aurescens were determined, and its physicochemical properties were examined. 1. The enzyme has been shown to be a glycoprotein containing mannose, glucose, glucosamine, and glucuronic acid (3:5:4:2). 2. Its molecular wieght was estimated to be 76,000 by gel filtration on Sephadex G-200, 75,000-80,000 by SDS disc electrophoresis, and 75,800 by sedimentation veolcity. No subunits were detected in the molecule. 3. The physicochemical properties determined include: sedimentation coefficient (s(o)20, w=5.14 S), diffusion constant (D(o)=6.09 X 10(-7) cm2/sec), frictional ratio (f:f(o)=1.19) and apparent partial specific volume (v=0.73 ml/g). 4. The optical rotatory dispersion and circular dichroism of the enzyme were investigated. The contents of alpha-helix and beta-structure of the enzyme were estimated to be 16 and 25%, respectively.     

Human kidney diamine oxidase: heterologous expression,purification, and characterization

Human kidney diamine oxidase has been overexpressed as a secreted enzyme under the control of a metallothionein promoter in Drosophila S2 cell culture. This represents the first heterologous overexpression and purification of a catalytically active, recombinant mammalian copper-containing amine oxidase. A rapid and highly efficient purification protocol using chromatography on heparin affinity, hydroxyapatite, and gel filtration media allows for the recovery of large quantities of the recombinant enzyme, which is judged to be greater than 98% homogenous by SDS/PAGE. The availability of large quantities of highly purified enzyme makes it now possible to investigate the spectroscopic, mechanistic, functional, and structural properties of this human enzyme at the molecular level. Visible absorption, circular dichroism, electron paramagnetic resonance, and resonance Raman spectroscopic results are presented. The recombinant enzyme contains the cofactors 2,4,5-trihydroxyphenylalaninequinone and copper at stoichiometries of up to 1.1 and 1.5 mol per mol homodimer, respectively. In addition, tightly bound and stoichiometric calcium ions were identified and proposed to occupy a second metal-binding site. The apparent molecular weight of the recombinant protein, determined by analytical ultracentrifugation, suggests 20-26% glycosylation by weight. Detailed kinetic studies indicate the preferred substrates (k(cat)/K(M)) of human diamine oxidase are, in order, histamine, 1-methylhistamine, and putrescine, with K(M) values of 2.8, 3.4, and 20 microM, respectively. These results, demonstrating the substrate preference for histamine and 1-methylhistamine, were unanticipated given the available literature. The pH dependence of k(cat) for putrescine oxidation gives two apparent p K(a) values at 6.0 and 8.2. Tissue-specific expression of the human diamine oxidase gene was investigated using an mRNA array. The relevance of this work to earlier work and the suggested physiological roles of the human enzyme are discussed.     

Crystallization and some properties of chondroitinase from Arthrobacter aurescens.

A strain of Arthrobacter aurescens which secretes a large amount of chondroitinase into a culture broth, was isolated from soil. The chondroitinase was purified 380-fold over culture broth in 24% yield and crystallized. Some properties of the purified enzyme were studied and described: thermal stability (below 45 degrees), pH stability (pH 4.9 to 7.4), optimum temperature (50 degrees), and optimum pH (pH 6.0). Chrondroitin sulfate A and C, chondroitin, and hyaluronic acid were split by the enzyme but dermatan sulfate could not be. The initial rates of enzymic degradation of chondroitin sulfate C, chondroitin, and hyaluronic acid were 1.1, 1.95, and 3.2, respectively, compared to that of chondroitin sulfate A. When the enzyme was allowed to act on chondroitin sulfate A and C, the reducing power and the ultraviolet absorption at 232 nm increased proportionally to the decrease in viscosity of the substrate solution. Finally these substrates were degraded to the extent of 100% to disaccharides. By the enzyme action the main products from chondroitin sulfate A and C were deta 4,5-unsaturated disaccharides, which were identified as 2-acetamido-2-deoxy-3-O-(Beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose and 2-acet-amido-2-deoxy-3-O-(Beta-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose by paper chromatography, ultraviolet absorption spectroscophy, and infrared spectroscopy. Thus it is suggested that the chondroitinase is a chondroitin sulfate A and C lyase, one of the hyaluronate lyases (EC 4.2.99.1).     

Production of L-tryptophan from D,L-5-indolylmethylhydantoin by resting cells of a mutant of Arthrobacter species (DSM 3747).

The reaction parameters and the stereospecificity of the enzymatic cleavage of D,L-5-indolylmethylhydantoin in producing L-tryptophan with resting cells of Arthrobacter sp. DSM 3747 were studied. When intact cells were tested, the optimal pH was between 8.5 and 9.0 and the optimal temperature was 50 degrees C. Both, L-N-carbamoylase and hydantoinase could be stabilized over 24 h at 30 and 40 degrees C by the addition of D,L-5-indolylmethylhydantoin. Furthermore, the hydantoinase was stable over 24 h at 50 degrees C by the addition of 0.5 mM Mn2+ ions. The treatment with sodium desoxycholate turned out to be successful in overcoming the poor availability of D,L-5-indolylmethylhydantoin for the cells. The optimal temperature with permeabilized cells decreased to 30 degrees C and therefore ensured a good enzyme stability. While the L-N-carbamoylase proved to be absolutely L-specific, the hydantoinase led to a mixture of enantiomers of N-carbamoyltryptophan. The produced D-N-carbamoyl-tryptophan caused an inhibition of the L-N-carbamoylase. The transformation yield from D,L-5-indolylmethylhydantoin always reached 100%.     

Purification and characterization of inulin fructotransferase (DFA III-forming) from Arthrobacter aurescens SK 8.001

The soil bacterium Arthrobacter aurescens SK 8.001 produces inulin fructotransferase (IFTase), and liquid chromatography-mass spectrometry (LC-MS) and carbon-13 nuclear magnetic resonance (13C NMR) analysis demonstrated that the main product of the enzyme was difructose anhydride III (DFA III). The IFTase was purified by ethanol precipitation, DEAE Sepharose Fast Flow, and Superdex 200 10/300 GL gel chromatography. Its molecular mass was estimated to be 40 kDa by SDS-PAGE and 35 kDa by gel filtration. The enzyme showed maximum activity at pH 5.5 and 60-70 °C, and retained 86.5% of its initial activity after incubation at 60 °C for 4 h. Chemical modification results suggested that a tryptophan residue is essential to enzyme activity. The N-terminal amino acid sequence was determined as AEGAKASPLNSPNVYDVT. The kinetic values, Km and Vmax, were estimated to be 0.52 mM and 0.3 μmol/ml min. Nystose was observed to be the smallest substrate for the produced IFTase. This IFTase provides a promising way to utilize inulin for the production of DFA III.     

Catabolic alanine racemase from Salmonella typhimurium: DNA sequence, enzyme purification, and characterization

The alanine racemase encoded by the Salmonella typhimurium dadB gene was purified to 90% homogeneity from an overproducing strain. At 37 degrees C the enzyme has a specific activity of 1400 units/mg (V max, L- to D-alanine). Active enzyme molecules are monomers of Mr 39 000 with one molecule of pyridoxal 5'-phosphate bound per subunit. The Km's for L- and D-alanine are 8.2 and 2.1 mM, respectively. Measurement of turnover numbers yielded the expected Keq value of 1.0. Determination of 22 of the 25 N-terminal amino acid residues of the purified polypeptide allowed localization of cloned DNA encoding the structural gene. Sequencing of subcloned DNA revealed that the dadB gene encodes a polypeptide of 356 amino acids whose calculated molecular weight (apoenzyme) was 39 044.     

Alanine racemase from Helicobacter pylori NCTC 11637: purification, characterization and gene cloning

The Helicobacter pylori NCTC 11637 alanine racemase gene, alr1, was cloned based on a putative alanine racemase gene, alr, of H. pylori 26695. The protein, Alr1, was purified to homogeneity from Escherichia coli MB2795 cells harboring the alr1 gene. The protein exclusively catalyzes the conversion of l-alanine to the d-isomer with K(m) and V(max) values of 100 mM and 909 mumol min(-1) mg(-1), respectively. The values are 16-fold higher than those for the reaction in the reverse direction. The molecular weight of Alr1 is 42,000 by SDS-PAGE, and 68,000 by gel-filtration analysis. The optimal pH and temperature are pH 8.3 and 37 degrees C, respectively, in good accordance with the characteristics shown by the alanine racemase purified from H. pylori NCTC 11637 cells. Pyridoxal 5'-phosphate was suggested to be the cofactor. The physiological function of Alr1 is discussed regarding energy production in the microbial cells.     

黑颈鹤粪便分离菌Arthrobacter sp. GN14 的α-半乳糖苷酶基因克隆、表达与酶学特性

【目的】克隆高原唯一珍惜鹤类——黑颈鹤粪便分离菌Arthrobacter sp.GN14的α-半乳糖苷酶基因agaAGN14,并对该酶进行序列分析、系统发育分析和重组酶的酶学特性分析。【方法】利用简并PCR和GCTAIL-PCR方法获得agaAGN14全长,并对其氨基酸序列(AgaAGN14)进行比对和neighbor-joining系统发育树的构建。将agaAGN14重组到载体pET-28a(+)中并转化到Escherichia coli BL21(DE3)中异源表达。利用组氨酸标签纯化重组α-半乳糖苷酶rAgaAGN14并进行酶学性质分析。【结果】agaAGN14全长2109 bp,GC含量66.8%,编码702个氨基酸(77.5 kDa)。AgaAGN14与数据库中序列的最高一致性为53.7%,与其余胃肠道环境α-半乳糖苷酶的一致性<43%。系统发育分析将AgaAGN14聚于具有催化域KWD和SDXXDXXXR的α-半乳糖苷酶分支,与土壤微生物来源α-半乳糖苷酶距离相对较近,而与其余胃肠道环境α-半乳糖苷酶距离相对较远。rAgaAGN14可水解pNPG、棉籽糖、密二糖、水苏糖、菜粕和棉籽粕,表观最适pH为6.5,在pH 6.0-pH 9.0的范围内稳定并维持50%以上的酶活性。rAgaAGN14的表观最适温度为45℃,在10℃、20℃和37℃内稳定并分别具有约28%、30%和80%的酶活。在45℃pH 6.5条件下,rAgaAGN14对pNPG的Km、Vmax和kcat分别为0.41 mmol/L、18.28μmol/min/mg和25.36 s-1。rAgaAGN14受Ag+、Hg2+及SDS抑制,受K+、Ca2+、Mn2+、Fe3+、Ni2+、Cu2+和β-mercaptoethanol部分抑制,受Co2+、Pb2+、Zn2+、Mg2+、Na+和EDTA的影响较小。【结论】首次报道从黑颈鹤粪便中分离到Arthrobacter菌,并对该属细菌α-半乳糖苷酶进行序列分析、系统发育分析、异源表达和重组酶的酶学特性分析。rAgaAGN14序列较新颖,其酶学特性可能是同时适应黑颈鹤肠道环境和高原淡水湿地环境的结果。     

Cloning, sequencing, heterologous expression, purification, and characterization of adenosylcobalamin-dependent D-ornithine aminomutase from Clostridium sticklandii

D-Ornithine aminomutase from Clostridium sticklandii catalyzes the reversible rearrangement of d-ornithine to (2R,4S)-2,4-diaminopentanoic acid. The two genes encoding d-ornithine aminomutase have been cloned, sequenced, and expressed in Escherichia coli. The oraS gene, which encodes a protein of 121 amino acid residues with M(r) 12,800, is situated upstream of the oraE gene, which encodes a protein of 753 amino acid residues with M(r) 82,900. The holoenzyme appears to comprise a alpha(2)beta(2)-heterotetramer. OraS shows no significant homology to other proteins in the Swiss-Prot data base. The deduced amino acid sequence of OraE includes a conserved base-off/histidine-on cobalamin-binding motif, DXHXXG. OraE was expressed in E. coli as inclusion bodies. Refolding experiments on OraE indicate that the interactions between OraS and OraE and the binding of either pyridoxal phosphate or adenosylcobalamin play important roles in refolding process. The K(m) values for d-ornithine, 5'-deoxyadenosylcobalamin (AdoCbl), and pyridoxal 5'-phosphate (PLP) are 44.5 +/- 2.8, 0.43 +/- 0.04, and 1.5 +/- 0.1 microm, respectively; the k(cat) is 6.3 +/- 0.1 s(-1). The reaction was absolutely dependent upon OraE, OraS, AdoCbl, PLP, and D-ornithine being present in the assay; no other cofactors were required. A red-shift in UV-visible absorption spectrum is observed when free adenosylcobinamide is bound by recombinant D-ornithine aminomutase and no significant change in spectrum when free adenosylcobinamide is bound by mutant OraE-H618G, demonstrating that the enzyme binds adenosylcobalamin in base-off/histidine-on mode.     

Collagenolytic serine-carboxyl proteinase from Alicyclobacillus sendaiensis strain NTAP-1: purification,characterization, gene cloning,and heterologous expression

Enzymatic degradation of collagen produces peptides, the collagen peptides, which show a variety of bioactivities of industrial interest. Alicyclobacillus sendaiensis strain NTAP-1, a slightly thermophilic, acidophilic bacterium, extracellularly produces a novel thermostable collagenolytic activity, which exhibits its optimum at the acidic region (pH 3.9) and is potentially applicable to the efficient production of such peptides. Here, we describe the purification to homogeneity, characterization, gene cloning, and heterologous expression of this enzyme, which we call ScpA. Purified ScpA is a monomeric, pepstatin-insensitive carboxyl proteinase with a molecular mass of 37 kDa which exhibited the highest reactivity toward collagen (type I, from a bovine Achilles tendon) among the macromolecular substrates examined. On the basis of the sequences of the peptides obtained by digestion of collagen with ScpA, the following synthetic peptides were designed as substrates for ScpA and kinetically analyzed: Phe-Gly-Pro-Ala*Gly-Pro-Ile-Gly (k(cat), 5.41 s(-1); K(m), 32 micro M) and Met-Gly-Pro-Arg*Gly-Phe-Pro-Gly-Ser (k(cat), 351 s(-1); K(m), 214 micro M), where the asterisks denote the scissile bonds. The cloned scpA gene encoded a protein of 553 amino acids with a calculated molecular mass of 57,167 Da. Heterologous expression of the scpA gene in the Escherichia coli cells yielded a mature 37-kDa species after a two-step proteolytic cleavage of the precursor protein. Sequencing of the scpA gene revealed that ScpA was a collagenolytic member of the serine-carboxyl proteinase family (the S53 family according to the MEROPS database), which is a recently identified proteinase family on the basis of crystallography results. Unexpectedly, ScpA was highly similar to a member of this family, kumamolysin, whose specificity toward macromolecular substrates has not been defined.     

A novel galactolipase from a green microalga Chlorella kessleri: purification,characterization, molecular cloning,and heterologous expression

We have identified an enzyme, galactolipase (ckGL), which hydrolyzes the acyl ester bond of galactolipids such as digalactosyldiacylglycerol (DGDG), in the microalga Chlorella kessleri. Following purification of the enzyme to electrophoretic homogeneity from cell-free extract, the maximum activity toward DGDG was observed at pH 6.5 and 37 °C. ckGL was Ca²⁺-dependent enzyme and displayed an apparent molecular mass of approx. 53 kDa on SDS-PAGE. The substrate specificity was in the order: DGDG (100%) > monogalactosyldiacylglycerol ≈ phosphatidylglycerol (~ 40%) > sulfoquinovosyldiacylglycerol (~ 20%); the enzyme exhibited almost no activity toward glycerides and other phospholipids. Gas chromatography analysis demonstrated that ckGL preferably hydrolyzed the sn-1 acyl ester bond in the substrates. The genomic DNA sequence (5.6 kb) containing the ckGL gene (designated glp1) was determined and the cDNA was cloned. glp1 was composed of 10 introns and 11 exons, and the 1608-bp full-length cDNA encoded a mature ckGL containing 475 amino acids (aa), with a presequence (60 aa) containing a potential chloroplast transit peptide. Recombinant functional ckGL was produced in Escherichia coli. Although the deduced aa sequence of ckGL contained the typical GXSXG motif of serine hydrolases together with conserved histidine and aspartate residues which would form part of the catalytic triad of α/β-hydrolases, ckGL showed no significant overall similarity with known lipases including GLs from Chlamydomonas reinhardtii and Aspergillus japonicus, indicating that ckGL is a novel GL. ckGL, with high specificity for DGDG, could be applicable to food processing as an enzyme capable of improving material textures.

     

Expression, purification, and characterization of alanine racemase from Pseudomonas putida YZ-26

Alanine racemase catalyzes the interconversion of d- and l-alanine and plays an important role in supplying d-alanine, a component of peptidoglycan biosynthesis, to most bacteria. Alanine racemase exists mostly in prokaryotes and is generally absent in higher eukaryotes; this makes it an attractive target for the design of new antibacterial drugs. Here, we present the cloning and characterization of a new gene-encoding alanine racemase from Pseudomonas putida YZ-26. An open reading frame (ORF) of 1,230 bp, encoding a protein of 410 amino acids with a calculated molecular weight of 44,217.3 Da, was cloned into modified vector pET32M to form the recombinant plasmid pET–alr. After introduction into E.coli BL21, the strain pET-alr/E.coli BL21 expressed His₆-tagged alanine racemase. The recombinant alanine racemase was efficiently purified to homogeneity using Ni²⁺–NTA and a gel filtration column, with 82.5% activity recovery. The amino acid sequence deduced from the alanine racemase gene revealed identity similarities of 97.0, 93, 23, and 22.0% with from P. putida F1, P. putida200, P. aeruginosa, and Salmonella typhimurium, respectively. The recombinant alanine racemase is a monomeric protein with a molecular mass of 43 kDa. The enzyme exhibited activity with l-alanine and l-isoleucine, and showed higher specificity for the former compared with the latter. The enzyme was stable from pH 7.0–11.0; its optimum pH was at 9.0. The optimum temperature for the enzyme was 37°C, and its activity was rapidly lost at temperatures above 40°C. Divalent metals, including Sr²⁺, Mn²⁺, Co²⁺, and Ni²⁺ obviously enhanced enzymatic activity, while the Cu²⁺ ion showed inhibitory effects.     

Extremely thermostable elongation factor G from Aquifex aeolicus: cloning, expression, purification, and characterization in a heterologous translation system

The fus gene of the translation factor G (EF-G) from the hyperthermophilic bacterium Aquifex aeolicus was cloned under control of a phage promoter and overexpressed in Escherichia coli with the T7 RNA polymerase system. A heat denaturation step at 95 degrees C was used to purify the protein from the cell extract. This approach simplified the chromatographic procedures and decreased the protein loss since most of Escherichia coli proteins were denatured and precipitated. Ten milligrams of the highly purified protein was isolated from 4 liters of induced culture. The overproduced EF-G was active in ribosome-dependent GTP hydrolysis and a poly(U)-directed polyphenylalanine translation system with E. coli 70S ribosomes. The method presented here might facilitate functional and structural studies of important components of the protein biosynthesis system.