This insightful overview by one of the most respected names in protein research discusses a broad array of the aspects involved with protein purification, including historical background, determining the purpose for purifying a particular protein, and actual methods and recommendations for purification procedures. Also covered are methods of analysis and detection, and techniques for characterizing a purified protein. Finally, a useful discussion of the equipment needed in a protein purification lab is provided.

This discussion unit presents information on classification of proteins according to shape, oligomeric structure, and function. In addition, strategies for purifying different types of proteins are discussed based on their location and associations within the cell. Specifically, critical issues are detailed for extracellular, cytoplasmic, and membrane-associated proteins, as well as for insoluble proteins. Finally, strategies for the purification of both soluble and insoluble (inclusion bodies) recombinantly expressed proteins are provided.

Several closely related, xylanolytic, thermophilic bacilli were isolated from local soils on xylose-containing minimal medium. On the basis of morphology and biochemical characteristics, one of the isolates, designated strain S1812T (T = type strain), was studied further. Strain S1812T was a xylanolytic, sporulating, Gram-positive, rod-shaped bacterium. Its Gram-positive nature was confirmed by electron microscopic examination of thin sections of the cells. The isolate was a thermophilic (optimum temperature for growth, 65 degrees C), facultative anaerobe that grew on a wide range of carbon sources including glucose, lactose, starch and xylose. It expressed high levels of both xylose isomerase and xylulokinase on xylose and also on glucose. The DNA G + C content was 44 mol%. rRNA gene sequence analysis placed strain S1812T in Bacillus cluster 5; it was more closely related to Saccharococcus thermophilus than to thermophilic Bacillus species. DNA-DNA hybridization also indicated its close relationship to S. thermophilus. Based on the evidence presented, it is proposed that strain S1812T be designated Saccharococcus caldoxylosilyticus sp. nov. Strain S1812T is the type strain (= ATCC 700356T = DSM 97-987T).

The groE homologous genes of the anaerobic thermophile Thermoanaerobacter sp. Rt8.G4 (TRt) have been isolated, sequenced and analysed. The TRt groES and groEL encode subunits of chaperonin 10 (Cpn10) and chaperonin 60 (Cpn60) of 94 and 541 amino acids, respectively, and are arranged in that order forming the open reading frames (ORFs) of a bicistronic operon. A controlling inverted repeat of chaperone expression (CIRCE) element lies between the consensus promoter of the operon and TRt groES. At optimum growth temperature (65°C) the chaperonins of TRt are expressed, but production of Cpn60 increases significantly following temperature increases of 3–10°C. Functionally intact recombinant TRt chaperonins were produced in Escherichia coli. However, owing to codon incompatibility, replacement of consecutive AGA codons in the gene encoding TRt Cpn60 was necessary for optimum expression in this heterologous host.

The effects of temperature on the kinetic parameters kcat and Km, for three isolates of the highly conserved monomeric enzyme 3-phosphoglycerate kinase (PGK), were investigated in detail using a rapid automated kinetics apparatus. PGK was purified from the thermophilic bacterium Thermoanaerobacter sp. Rt8.G4 (optimum growth temperature 68 degrees C), the mesophile Zymomonas mobilis (optimum growth temperature 32 degrees C) and a second, unidentified, soil mesophile designated unid A (optimum growth temperature 27 degrees C). The kinetic behaviour with temperature of each PGK preparation was distinct, despite the conserved nature of the enzyme. The kcat values increased with temperature, but not as rapidly exponentially, as might be expected from the Arrhenius equation. Maximum kcat values were at much higher temperatures than the optimum growth temperatures for the mesophiles, but for the thermophile the temperature of maximum kcat was close to its optimum growth temperature. Km values were in general nearly constant through the lower temperature ranges, but increased substantially as the optimum temperature (highest kcat) was passed. Thermal irreversible denaturation of the PGK proteins was also investigated by measuring loss of activity over time. In a dilute buffer, Arrhenius plots for denaturation were linear, and the calculated apparent energy of activation (Eact) for denaturation for the thermophilic PGK was 600 kJ.mol-1, whereas for the mesophilic enzymes the values were 200-250 kJ.mol-1. In the presence of substrates, a considerable stabilization occurred, and in the case of the Z. mobilis enzyme, the apparent Eact was increased to 480 kJ.mol-1. A theoretical explanation for these observations is presented. Comparing the kinetics data with irreversible denaturation rates determined at relevant temperatures, it was clear that kcat values reached a maximum, and then decreased with higher temperature before irreversible denaturation had any significant influence.

The second enzyme of the Entner-Doudoroff glycolytic pathway in Zymomonas mobilis, glucose-6-phosphate dehydrogenase, has been found to be inhibited by phosphoenolpyruvate (PEP). In the presence of PEP levels in the micromolar range, the response of the enzyme to glucose 6-phosphate concentration becomes sigmoidal, with a Hill coefficient up to 2. At low ionic strength in the absence of PEP, the response to glucose 6-phosphate concentration is Michaelis-Menten, but at physiological ionic strength and pH, a Hill coefficient of 1.3 to 1.4 was found even in the absence of PEP. Km values for NAD+ and NADP+ are also ionic-strength-dependent, increasing rapidly as salt concentration increases. Some sigmoidicity was also observed for NAD+ in the presence of PEP at low glucose 6-phosphate concentrations. The results can be interpreted in a Monod-Wyman-Changeux model, in which glucose 6-phosphate binds principally to the R-state, PEP to the T-state, and NAD+ to both states. These observations are clearly physiologically significant, and provide an explanation for the control of the balance between glycolytic throughput and ATP consumption in Z. mobilis.

The requirements for divinylsulfone (DVS)-based gels to act as thiophilic adsorbents, binding immunoglobulins in a salt-dependent manner have been examined. No differences in protein binding were observed for a DVS-activated gel reacted with mercaptoethanol (the T-gel), or for the same gel treated at high pH to hydrolyse the active groups and/or allow the formation of cross-links within the matrix, indicating that an O atom may be substituted for the thioether without affecting the thiophilic interactions. Extending the time of the activation reaction between DVS and the matrix results in increased amounts of sulfone attached to the gel, but decreased levels of active vinyl groups. When coupled to mercaptoethanol, these adsorbents bound more IgG than gels activated for shorter periods. This provides a convenient method to prepare thiophilic adsorbents of high capacity while minimising the amount of DVS used.The immobilised vinylsulfone must be linked to an electron donating atom for IgG to bind. When the vinyl was instead reduced with sodium borohydride, protein binding was decreased. No IgG bound to amine-coupled DVS-activated adsorbents, perhaps due to an overall positive charge on these gels at pH 7.4.The binding of human IgG to the adsorbents is dependent on ligand density, with little protein binding to gels having less than 16 mu mol sulfone per mi. The binding increased with the ligand density above this level, with more than 25 mg IgG binding per mi to an adsorbent having 114 mu mol sulfone per mi. The lack of binding at low ligand densities would be expected if the IgG must interact with two or more sulfone ligands to be retained on the adsorbent. (C) 1997 Elsevier Science B.V.

Random mutagenesis of the gene encodingZymomonas mobilisalcohol dehydrogenase-2 has enabled isolation of variants of the enzyme that have substrate specificities different from that of the wild-type enzyme. After amino acids responsible for the changes were identified, directed mutation at these sites was also carried out. Variants that are active on butanol have been investigated in detail. Changes at residue 161 and other changes at residues 155 and 165 cause enhanced activity with longer-chain alcohols. The 165 change also induces a marked alcohol-activation phenomenon that is observed not only with ethanol, but also with a nonsubstrate alcohol, 2-propanol, and with low concentrations of Triton X-100. These alterations to the alcohol binding pocket mainly introduce larger, more hydrophobic residues, suggesting that it is not the size but the hydrophobicity of the pocket that affects the substrate specificity. Variants active with NADP were isolated, and, as with similar variants of the yeast enzyme, they were found to have an Asp residue replaced by a neutral amino acid. However, unlike the yeast examples in which the affinity was substantially reduced, the affinity for NAD+in these variants was little changed, and the affinity for NADP+was higher than that for NAD+. As this enzyme is naturally ferrous ion-activated, and inactive with zinc, attempts were made to find variants that had activity with zinc. One was found, but the screening method also isolated other variants with altered metal ion preferences due to a mutation affecting amino acid 330.

As a first step in the development of a reporter system for gene expression in halophilic archaea, a β-galactosidase was purified 140-fold from Haloferax alicantei (previously phenon K, strain Aa2.2). An overproducing mutant was first isolated by UV mutagenesis and screening on agar plates containing X-Gal substrate. Cytoplasmic extracts of the mutant contained 25-fold higher enzyme levels than the parent. Purification of the active enzyme was greatly facilitated by the ability of sorbitol to stabilise enzyme activity in the absence of salt, which allowed conventional purification methods (e.g., ion-exchange chromatography) to be utilised. The enzyme was optimally active at 4 M NaCl and was estimated to be 180±20 kDa in size, consisting of two monomers (each 78±3 kDa). It cleaves several different β-galactoside substrates such as ONP-Gal, X-Gal and lactulose, but not lactose, and also has β-d-fucosidase activity. No β-glucosidase, β-arabinosidase or β-xylosidase activity could be detected. The amino-acid sequence at the N-terminus and of four proteolytic products has been determined.

Biotechnology and Applied BiochemistryVolume 23, Issue 3 p. 197-204 Protein purification in the nineties Scopes RK, Scopes RKSearch for more papers by this author Scopes RK, Scopes RKSearch for more papers by this author First published: June 1996 https://doi.org/10.1111/j.1470-8744.1996.tb00375.xAbout ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume23, Issue3June 1996Pages 197-204 RelatedInformation

An assay has been developed for measuring the number of reactive groups on gels activated for use in affinity chromatography with epichlorohydrin or divinylsulfone. The activated gel is reacted with mercaptoacetic acid and the number of acidic groups introduced is determined by titration. The results obtained are in good agreement with those obtained by elemental analysis and are significantly higher than those obtained by reaction with sodium thiosulfate. Using this assay, the epichlorohydrin and divinylsulfone activation reactions were optimised so that, by varying the amount of activating reagent and time of the reaction, Sepharose CL-4B can be activated in a controlled manner, to between 2 and 34 μmol active groups per ml of gel with epichlorohydrin, and to between 2 and 55 μmol active groups per ml of gel with divinylsulfone. Activation at pH 12 was found to be optimal for divinylsulfone activation. The stability of these activated gels to incubation for 40 h over the pH range 2–14 was examined, as was the stability to long-term storage at 4°C.

Journal of Molecular RecognitionVolume 9, Issue 5-6 p. 305-308 Critical Review Screening for protein purification: Is there an optimum adsorbent for every protein? Robert K. Scopes, Robert K. Scopes School of Biochemistry, La Trobe University, Bundoora, VIC 3083, AustraliaSearch for more papers by this author Robert K. Scopes, Robert K. Scopes School of Biochemistry, La Trobe University, Bundoora, VIC 3083, AustraliaSearch for more papers by this author First published: 4th Quarter 1996 https://doi.org/10.1002/(SICI)1099-1352(199634/12)9:5/6<305::AID-JMR270>3.0.CO;2-QCitations: 5AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume9, Issue5-6Special Issue: 11th International Symposium on Affinity Chromatography and Biological Recognition4th Quarter 1996Pages 305-308 RelatedInformation

Annals of the New York Academy of SciencesVolume 799, Issue 1 p. 752-756 Inactivation of Glucose-Fructose Oxidoreductase from Zymomonas mobilis during Its Catalytic Actionsa MONIKA FÜRLINGE, MONIKA FÜRLINGE Division of Biochemical Engineering Institute of Food Technology Universität für Bodenkultur Wien (BOKU) 1190 Vienna, AustriaSearch for more papers by this authorBERNDN IDETZKY, BERNDN IDETZKY Division of Biochemical Engineering Institute of Food Technology Universität für Bodenkultur Wien (BOKU) 1190 Vienna, Austria To whom all correspondence should be addressed.Search for more papers by this authorROBERT K. SCOPES, ROBERT K. SCOPES Department of Biochemistry La Trobe University Bundoora, Victoria 3083, AustraliaSearch for more papers by this authorDIETMAR HALTRICH, DIETMAR HALTRICH Division of Biochemical Engineering Institute of Food Technology Universität für Bodenkultur Wien (BOKU) 1190 Vienna, AustriaSearch for more papers by this authorKLAUS D. KULBE, KLAUS D. KULBE Division of Biochemical Engineering Institute of Food Technology Universität für Bodenkultur Wien (BOKU) 1190 Vienna, AustriaSearch for more papers by this author MONIKA FÜRLINGE, MONIKA FÜRLINGE Division of Biochemical Engineering Institute of Food Technology Universität für Bodenkultur Wien (BOKU) 1190 Vienna, AustriaSearch for more papers by this authorBERNDN IDETZKY, BERNDN IDETZKY Division of Biochemical Engineering Institute of Food Technology Universität für Bodenkultur Wien (BOKU) 1190 Vienna, Austria To whom all correspondence should be addressed.Search for more papers by this authorROBERT K. SCOPES, ROBERT K. SCOPES Department of Biochemistry La Trobe University Bundoora, Victoria 3083, AustraliaSearch for more papers by this authorDIETMAR HALTRICH, DIETMAR HALTRICH Division of Biochemical Engineering Institute of Food Technology Universität für Bodenkultur Wien (BOKU) 1190 Vienna, AustriaSearch for more papers by this authorKLAUS D. KULBE, KLAUS D. KULBE Division of Biochemical Engineering Institute of Food Technology Universität für Bodenkultur Wien (BOKU) 1190 Vienna, AustriaSearch for more papers by this author First published: October 1996 https://doi.org/10.1111/j.1749-6632.1996.tb33286.x a This work was supported financially by the Jubiliäumsfonds der Österreichischen National-bank (Grant No. P 5089). AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Volume799, Issue1Enzyme Engineering XIIIOctober 1996Pages 752-756 RelatedInformation

The iron-dependent alcohol dehydrogenase from Zymomonas mobilis has been crystallized in a form suitable for X-ray diffraction studies. The crystals grew in hanging drops by vapor diffusion, equilibrating with a solution comprising 25-27% methoxypolyethylene glycol 5000 and 1 mM Co(2+) in a 0.2 M succinic acid/potassium hydroxide buffer at pH 5.5-5.7 at 281 K. Crystals are tetragonal, P4(1)22 (or P4(3)22), with unit-cell dimensions a = b = 125.7, c = 248.1 A. Four molecules comprise the asymmetric unit, and a self-rotation function indicates twofold local symmetry perpendicular to the unique axis and 15 degrees from a crystallographic twofold axis. Diffraction data to 3.0 A have been collected.

The gene encoding fructokinase (EC 2.7.1.4) from Zymomonas mobilis has been expressed at high level in Escherichia coli by modifying the ribosome binding site using the polymerase chain reaction. A simple two-step purification from extracts of the recombinant cells results in highly purified enzyme suitable for use in fructose determination. Using the polymerase chain reaction in mutagenic conditions, a variant of fructokinase was isolated which was more thermostable than the wild type, taking the 30 min half-life from 70.1 to 72.4 degrees C. The purified thermostable variant had the same specific activity as the wild type. Sequencing of the variant indicated that only one amino acid was changed, with Ser 69 becoming Ala. Searches of the mutant libraries for variants that were (a) active with glucose or (b) had reduced inhibition by glucose were unsuccessful.

BACKGROUND:The organism Zymomonas mobilis occurs naturally in sugar-rich environments. To protect the bacterium against osmotic shock, the periplasmic enzyme glucose-fructose oxidoreductase (GFOR) produces the compatible, solute sorbitol by reduction of fructose, coupled with the oxidation of glucose to gluconolactone. Hence, Z mobilis can tolerate high concentrations of sugars and this property may be useful in the development of an efficient microbial process for ethanol production. Each enzyme subunit contains tightly associated NADP which is not released during the catalytic cycle. RESULTS:The structure of GFOR was determined by X-ray crystallography at 2.7 A resolution. Each subunit of the tetrameric enzyme comprises two domains, a classical dinucleotide-binding domain, and a C-terminal domain based on a predominantly antiparallel nine-stranded beta sheet. In the tetramer, the subunits associate to form two extended 18-stranded beta sheets, which pack against each other in a face to face fashion, creating an extensive interface at the core of the tetramer. An N-terminal arm from each subunit wraps around the dinucleotide-binding domain of an adjacent subunit, covering the adenine ring of NADP. CONCLUSIONS:In GFOR, the NADP is found associated with a classical dinucleotide-binding domain in a conventional fashion. The NADP is effectively buried in the protein-subunit interior as a result of interactions with the N-terminal arm from an adjacent subunit in the tetramer, and with a short helix from the C-terminal domain of the protein. This accounts for NADP's inability to dissociate. The N-terminal arm may also contribute to stabilization of the tetramer. The enzyme has an unexpected structural similarity with the cytoplasmic enzyme glucose-6-phosphate dehydrogenase (G6PD). We hypothesize that both enzymes have diverged from a common ancestor. The mechanism of catalysis is still unclear, but we have identified a conserved structural motif (Glu-Lys-Pro) in the active site of GFOR and G6PD that may be important for catalysis.

Protein purification flow charts are presented to give a broad outline of the methods used for different types of proteins. They cannot give any detail, as the process appropriate for each protein will have its own variations at each stage. In most cases, the first stage is to obtain a solution containing the desired protein, after which it can be fractionated by various separation techniques. This unit provides charts tailored for purification of soluble and insoluble recombinant and non-recombinant proteins, as well as membrane-associated proteins.

A detailed kinetic analysis of glucokinase EC 2.7.1.2 from Zymomonas mobilis has been carried out. This enzyme has an absolute requirement for inorganic phosphate as activator, and the kinetic behaviour can be interpreted as a steady-state ordered mechanism in which glucose is the first substrate. Values for each of the kinetic constants have been obtained for the conditions I = 0.12, 30 degrees C, and pH 7.0. Direct binding studies have confirmed that ATP does not bind to the enzyme without glucose present. Phosphate does not affect ATP binding to the enzyme-glucose complex; when saturated with both ATP and glucose, the dissociation constant for phosphate (determined kinetically) is 0.045 mM. When saturated with the other substrate and phosphate, the Km values for glucose and MgATP are 0.095 mM and 0.19 mM, respectively. The ionic form of phosphate is not important, as the apparent Km for phosphate did not change significantly over the pH range 6.4 to 7.5. Raising the temperature increased Vmax at the high rate of 10% per degree, which correlates well with the fermentation rates between 20 and 30 degrees C, giving further support to the concept that glucokinase is the rate-controlling enzyme in Z. mobilis glucose fermentation.