Journal of Chromatography B (v.786, #1-2)

Preface (1-2).

Purification of the proline-rich homeodomain protein by Amy J. Butcher; Kevin Gaston; Padma-Sheela Jayaraman (3-6).
The proline-rich homeodomain protein (PRH), also known as Hex, is a transcriptional repressor expressed in a variety of cell types. The PRH protein contains a proline-rich N-terminal domain that can repress transcription when attached to a heterologous DNA binding domain, a central homeodomain that mediates sequence-specific DNA binding, and an acidic C-terminal domain of unknown function. Although individual domains of PRH have been expressed in bacterial cells as GST- and histidine-tagged fusion proteins, attempts to express and purify the full-length protein have met with little success. Here we describe the purification of a histidine-tagged full-length PRH fusion protein. The protein described here will allow us to determine the mechanisms whereby PRH represses transcription.
Keywords: Proline-rich homeodomain protein;

Purification of human alpha-l-fucosidase precursor expressed in Escherichia coli as a glutathione S-transferase fusion protein by Alejandro de Carlos; Dolores Montenegro; Ana Alonso-Rodrı́guez; Marı́a Páez de la Cadena; Francisco Javier Rodrı́guez-Berrocal; Vicenta Soledad Martı́nez-Zorzano (7-15).
Alpha-l-fucosidase (FUC) is a glycosidase involved in the degradation of fucose-containing glycoconjugates. A cDNA representing the complete sequence of human FUC was inserted into the prokaryotic expression vector pGEX-2T. High levels of the glutathione S-transferase (GST) fusion protein were detected in Escherichia coli cells after induction with isopropyl thio-beta-d-galactopyranoside. The GST-FUC protein was mostly found as inclusion bodies and attempts to optimise its expression as a soluble form were unsuccessful. Nevertheless, the recombinant protein was purified by affinity chromatography on glutathione-sepharose and its fucosidase activity was characterised. After thrombin cleavage of the GST tag, the FUC precursor protein was purified by electro-elution.
Keywords: α-l-Fucosidase precursor; Glutathione S-transferase fusion protein;

Site directed mutagenesis of Cys17→Ser17 form of recombinant human granulocyte colony stimulating factor (rhG-CSF C17S) for sequential replacing of surface His43 and His52 with alanine was used to identify residues critical for the protein interaction with metal ions, in particular Ni2+ chelated by dye Light Resistant Yellow 2 KT (LR Yellow 2KT)–polyethyleneglycol (PEG), and refolding after partitioning of inclusion bodies in aqueous two-phase systems. Strong binding of rhG-CSF (C17S) to PEG–LR Yellow 2KT–Cu(II) complex allowed for the adoption of affinity chromatography on Sepharose–LR Yellow 2KT–Cu(II) that appeared to be essential for the rapid isolation of mutated forms of rhG-CSF. Efficiency of that purification stage is exemplified by isolation of rhG-CSF (C17S, H43A) and rhG-CSF (C17S, H43A, H52A) mutants in correctly folded and highly purified state. Affinity partitioning of rhG-CSF histidine mutants was studied in aqueous two-phase systems containing Cu(II), Ni(II) and Hg(II) chelated by LR Yellow 2KT–PEG at pH 7.0 and Cu(II)—at pH 5.0. It was determined, that affinity of rhG-CSF mutants for metal ions decreased in the order of C17S>C17S, H43A>C17S, H43A, H52A for Cu(II), and C17S=C17S, H43A>C17S, H43A, H52A for Ni(II) ions, while affinity of all rhG-CSF mutants for Hg(II) ions was of the same order of magnitude. Influence of His43 and His52 mutation on protein refolding was studied by partitioning of the respective inclusion body extract in aqueous two-phase systems containing Ni(II) and Hg(II) ions. Data on rhG-CSF histidine mutant partitioning and refolding indicated, that His52 mutation is crucial for the strength of protein interaction with chelated Ni(II) ions and refolding efficiency.
Keywords: Proteins; Metal chelates;

ShlB from Serratia marcescens was isolated and purified from a porin-deficient Escherichia coli BL21 strain using a combination of detergent extraction, affinity and ion-exchange chromatography. An internal histidine affinity tag was introduced that did not interfere with activity. At each stage of the purification scheme biological activity of the ShlB protein was assessed. Using this scheme, several His6-tagged mutants of ShlB were purified to electrophoretic homogeneity.
Keywords: Outer membrane protein;

Purification of recombinantly expressed human cluster determinant 4 cytoplasmic domain by Andrea Preusser; Gesa Jonas; Dieter Willbold (39-44).
A DNA fragment coding for the human CD4 cytoplasmic domain (residues 394–433) was cloned into the pET15b expression vector. The resulting plasmid was used for synthesis of the polyhistidine-tagged 5·103 M r CD4 peptide in Escherichia coli BL21(DE3)Star. The CD4 cytoplasmic domain was purified under denaturing and reducing conditions by a two-step procedure using immobilized metal affinity chromatography and gel permeation chromatography. The purified CD4 cytoplasmic domain is soluble and functional without any specific refolding steps. The yield of the described purification procedure was ∼5 mg peptide per liter culture volume.
Keywords: Cluster determinant 4;

Purification of recombinant BtpA and Ycf3, proteins involved in membrane protein biogenesis in Synechocystis PCC 6803 by Tatjana M.E. Schwabe; Kirsten Gloddek; Daniela Schluesener; Jochen Kruip (45-59).
The gene products Ycf3 (hypothetical chloroplast open reading frame) and BtpA (biogenesis of thylakoid protein) are thought to be involved in the biogenesis of the membrane protein complex photosystem I (PSI) from Synechocystis PCC 6803. PSI consists of 12 different subunits and binds more than 100 cofactors, making it a model protein to study different aspects of membrane protein biogenesis. For a detailed biophysical characterization of Ycf3 and BtpA pure proteins must be available in sufficient quantities. Therefore we cloned the corresponding genes into expression vectors. To facilitate purification we created His-tagged versions of Ycf3 and BtpA in addition to the unmodified forms. Immobilized metal affinity chromatography (IMAC) yielded His-tagged proteins which were used for the production of antibodies. Purification strategies for non-tagged proteins could also be established: Ycf3 could be purified in soluble form using a two-step purification in which ammonium sulfate precipitation was combined with anion-exchange chromatography (IEC). BtpA had to be purified from inclusion bodies by two-consecutive IEC steps under denaturing conditions. An optimized refolding protocol was established that yielded pure BtpA. In all cases, MALDI-TOF peptide mass fingerprinting (PMF) was used to confirm protein identity. Initially, size exclusion chromatography and CD-spectroscopy were used for biophysical characterization of the proteins. Both Ycf3 and BtpA show homo-oligomerization in vitro. In summary, purification protocols for Ycf3 and BtpA have been designed that yield pure proteins which can be used to probe the molecular function of these proteins for membrane protein biogenesis.
Keywords: Membrane proteins; Photosystem I;

Expression and purification of Plasmodium falciparum MSP-142: A malaria vaccine candidate by Christian Epp; Christian W Kauth; Hermann Bujard; Rolf Lutz (61-72).
The C-terminal 42·103 Da portion of the merozoite surface protein (MSP-1) of the human malaria parasite Plasmodium falciparum is of interest, not only because it may constitute an essential part of a future anti-malaria vaccine, but also due to its role during the infection of erythrocytes by the parasite. We have cloned and expressed two synthetic DNA sequences encoding the two prototypic MSP-142 variants in E. coli. When over-produced, both proteins form insoluble aggregates which were isolated in high purity and yield. After solubilisation and refolding in vitro, both proteins were purified to homogeneity by a three-step procedure applying Ni-chelate, size exclusion and immuno-affinity chromatography. After purification, both proteins meet key criteria of preparations for clinical use. First, conformational studies suggest proper folding of the proteins, particularly in the region containing two EGF-like domains. Polyclonal serum raised against E. coli produced MSP-142 recognizes native MSP-1 in Plasmodium infected erythrocytes as shown by immunofluorescence.
Keywords: MSP-142;

The development of thrombin inhibitors could provide invaluable progress for antithrombotic therapy. In this paper, we report the cloning, purification and biochemical characterization of dipetarudin, a chimeric thrombin inhibitor composed of the N-terminal head structure of dipetalogastin II, the strongest inhibitor from the assassin bug Dipetalogaster maximus, and the exosite 1 blocking segment of hirudin, connected through a five glycine linker. The cloning of dipetarudin was performed by a simple method which had not been used previously to clone chimeras. Biochemical characterization of dipetarudin revealed that it is a slow, tight-binding inhibitor with a molecular mass (M r=7560) and a thrombin inhibitory activity (K i=446 fM) comparable to r-hirudin.
Keywords: Dipetarudin; Thrombin inhibitor;

We have identified nine cyclophilins encoded in the genome of the fission yeast Schizosaccharomyces pombe (Sp). Cyclophilin 3 is an orthologue of hUSA-CyP, which is associated with Prp4/Prp3 in the [U4/U6·U5] snRNP complex and Prp18, both of which are components of the pre-mRNA splicing machinery. PPIase assays have shown SpCyp3 and hUSA-CyP to have comparable activity and substrate specificity, but SpCyp3 has a reduced sensitivity to CsA correlating with a difference in the catalytic site. Prp3, Prp4 and Prp18 proteins exist in S. pombe and nuclear localisation of SpCyp3 has been shown, indicating conservation of function between hUSA-CyP and SpCyp3.
Keywords: Cyclophilin; Peptidyl-prolyl cis/trans isomerase (PPIase);

The strategy described in this paper provides a novel approach for recombinant expression of heterodimeric proteins, and is especially suitable for the production of proteins whose characteristics lead to aggregation in E. coli expression systems. Pheromaxein, a steroid-binding protein isolated from boar saliva, is a heterodimeric protein consisting of 10×103 rel. mol. mass units (pheromaxein A) and 8×103 rel. mol. mass units (pheromaxein C) subunits. Expression of pheromaxein subunits in E. coli resulted in extensive insoluble aggregation. The difficulty faced in obtaining soluble recombinant pheromaxein subunits was clearly evident when native pheromaxein immediately formed aggregates when it was separated into its individual subunits. An increase in soluble pheromaxein expression in E. coli was obtained when the subunits were expressed as fusion proteins with thioredoxin. Pheromaxein genes were inserted separately into pET32a+ vectors at the NcoI site, resulting in thioredoxin, S·Tag™ and His·Tag™ coding regions being upstream of the inserted gene. Soluble pheromaxein A–thioredoxin (pheroA/trx) and pheromaxein C–thioredoxin (pheroC/trx) fusions were purified to homogeneity, using a laboratory scale S-protein agarose affinity column. Cleavage of thioredoxin under normal conditions was not feasible due to the extensive aggregation problems experienced when pheromaxein subunits exist separately. PheroA/trx and pheroC/trx were therefore mixed together and cleaved from thioredoxin simultaneously so that pheromaxein subunits were given an instant opportunity to associate under oxido-shuffling conditions. The glutathione oxido-shuffling system allowed the disulphide bridges between pheromaxein A and C to rearrange until the correct native structure was formed. This novel approach combines affinity purification with a coupled fusion protein-cleavage and refolding technique.
Keywords: Recombinant heterodimeric protein;

Heat shock proteins with a molecular mass of 70 000 (Hsp70s) are a ubiquitous class of ATP-dependent molecular chaperones involved in the folding of cellular proteins. Sequencing the entire genome of Saccharomyces cerevisiae revealed 14 different genes for Hsp70 proteins in different cellular compartments. Among these 14 Hsp70s, the subclass of Ssa (Ssa1p–Ssa4p) is abundant and essential in the cytosol. Since high yield expression of cytoplasmic Ssa1p is inefficient in Saccharomyces cerevisiae and recombinant expression in E. coli yields low protein levels, we chose Pichia pastoris as the recombinant expression system. In Pichia pastoris, expression levels of Ssa1p are high and Ssa1p is soluble and correctly folded. Also, we present a new protocol for purification of Ssa1p. Previously described purifications include ATP-agarose chromatography leading to Ssa1p partially complexed with ATP. Our optimized purification protocol follows the CiPP strategy (capture, intermediate purification, polishing) avoiding ATP-agarose chromatography, which allows detailed studies on the ATP-dependent Hsp70 functions. We obtained Ssa1p in high purity and 400 times higher quantity compared to previous studies.
Keywords: Ssa1p; Hsp70; ATPase;

Cloning, expression and purification of three Chaperonin 60 homologues by Maria Maguire; Anthony R.M Coates; Brian Henderson (117-125).
The Chaperonin 60 (Cpn60) proteins have, in addition to their well-known functions of protein folding and protection, a range of intercellular signalling activities. As part of a study to investigate the biological activity of the Cpn60 proteins, particularly from pathogenic organisms, we have cloned and expressed three Cpn60 proteins from Homo sapiens, Helicobacter pylori and Chlamydia pneumoniae. The Cpn60 proteins were purified to apparent homogeneity using a combination of nickel column affinity chromatography and Reactive Red dye affinity columns. Insoluble protein was solubilised using 8 M urea and then re-folded on the nickel column by stepwise removal of the urea. The immunostimulant LPS was removed by addition of the antibiotic polymyxin B as part of the purification process.
Keywords: Chaperonin 60 proteins;

Citrate synthase (CS) is a dimeric, mitochondrial protein, composed of two identical subunits (M r 48 969 each). The nuclear-encoded α-helical protein is imported into mitochondria post-translationally where it catalyses the first step of the citric cycle. Furthermore, the pathway of thermal unfolding as well as the folding pathway was studied extensively, making CS a well-suited substrate protein for studying chaperone function. In chaperone research the quality of the substrate proteins is essential to guaranty the reproducibility of the results. In this context, we here describe the GroE-enhanced recombinant expression and purification of CS. CS was expressed in E. coli by using an arabinose regulated T7 promotor. Under standard expression conditions only insoluble, inactive CS was detected. Interestingly, the expression of soluble and active CS was possible when GroEL/GroES was co-expressed. Furthermore, a shift to lower expression temperatures increased the amount of soluble, active CS. We describe for the first time, the purification of CS in soluble and active form by following a CiPP strategy (capture, intermediate purification, polishing). After the initial capturing step on DEAE-Sephacel the protein was further purified on a Q-Sepharose column. After these two steps of anion-exchange chromatography a final size-exclusion chromatography step on a Superdex 75-pg column yields CS with a purity over 99%. Using this expression and purification strategy 1 mg CS per g E. coli wet weight were purified.
Keywords: Citrate synthase; Enzymes;

In vitro cell free synthesis of human manganese superoxide dismutase with the RTS 500 system by Michael Kleines; Martin Nellessen1 ; Lars Schaade; Klaus Ritter (137-142).
Reduced activity of manganese superoxide dismutase (MnSOD) is the basis of several pathologic features and complications occurring in the course of infectious mononucleosis. In order to supply future research with easily accessible enzyme, an in vitro protocol was developed based on the RTS 500 system and an overexpression vector. Translation of MnSOD monomers could be detected by SDS–PAGE, and assembly of the active homotetramer by native PAGE. Enzyme activity was successfully shown by in gel activity tests and enzyme assays. With 15 μg of DNA, 2.45 μkat were generated. The purification of MnSOD was performed by chromatography applying the His-tag technology. In SDS–PAGE of the eluate, a band showed up at M r 25 000.
Keywords: Enzymes; Manganese superoxide dismutase;

Simplified procedure to recover recombinant antigenized secretory IgA to be used as a vaccine vector by Laurent I. Favre; François Spertini; Blaise Corthésy (143-151).
Induced protection mechanisms at mucosal surfaces involve secretory IgA (SIgA), a complex structure made of polymeric–dimeric IgA (IgAp/d) antibody associated with secretory component (SC). SIgA can adhere to M cells of the intestinal and nasal epithelia, are transported across these latter, and are thus available to the immune cells underlying the epithelia. This property makes SIgA suitable as potential mucosal vaccine delivery vector. It remains that production and purification of SIgA is a complex task since IgAp/d and SC are naturally synthesized by two different cell types. Furthermore, only IgAp/d are capable to associate with SC. Thus, we sought to separate IgAp/d and monomeric IgA (IgAm) antibodies secreted by hybridoma cells in CELLine bioreactors. To this aim, we connected together two 1-m long columns filled with Sephacryl S-300 beads and placed them under the control of a automatized chromatographic system. In parallel, we produced recombinant antigenized human SC (ra-hSC) in Chinese hamster ovary (CHO) cells adapted to suspension culture in CELLine bioreactors. To avoid intermediate purification of ra-hSC, culture supernatants (SN) containing this latter were combined with purified IgAp/d, and the recombinant antigenized SIgA (raSIgA) complex was resolved on a 1-m long column filled with Superdex 200 beads. Biochemical characterization based on SDS–PAGE, silver staining, immunodetection and enzyme-linked immunosorbent assay (ELISA) indicates that highly purified raSIgA can be recovered using this simple two-step procedure. Such preparations are currently used to immunize mice to induce mucosal and systemic responses.
Keywords: Immunoglobulin A; Recombinant protein;

In this report, we describe a two-step chromatographic procedure for the purification of His-tag EGFP by immobilized metal affinity expanded bed adsorption (IMAEBA) as the capture step and size exclusion chromatography as the polishing step. The use of proteins including a histidine-tag facilitates their subsequent purification after expression in many microorganisms. This meets the needs of scientific researchers as well as industrialists in purifying recombinant proteins. The procedure described allowed the obtention of 230 mg pure EGFP from 1 l simple batch culture with a recovery of 90%.
Keywords: Enhanced green fluorescent protein;

Optimizing expression and purification from cell culture medium of trispecific recombinant antibody derivatives by An Willems; Jannick Leoen; Steve Schoonooghe; Johan Grooten; Nico Mertens (161-176).
Antibody fragments offer the possibility to build multifunctional manifolds tailored to meet a large variety of needs. We optimized the production of a manifold consisting of one (bibody) or two (tribody) single-chain variable fragments coupled to the C-terminus of Fab chains. Different strong mammalian promoters were compared and the influence of expression media on production and recovery was investigated. Since the physical and chemical nature of these molecules largely depends on the nature of the antibody building blocks incorporated, a generally applicable process for the purification of recombinant antibody derivatives from serum containing mammalian cell culture medium was designed. To this end we compared protein L, hydroxyapatite, immobilized metal affinity chromatography, cation-exchange chromatography and hydrophobic charge induction chromatography.
Keywords: Bispecific antibodies; Fab fragment; Single-chain variable fragments;

In this study, we compared two gene fusion expression strategies using two rare codon genes (Ssh10b and MtGrxM) from archaea as a model system. Both genes can be highly expressed as N- or C-terminal fusion partners to GST or the intein/chitin-binding tag. However, the fusion protein with intein tag could not be cleaved, even under stringent conditions, possibly due to steric hindrance, thus preventing further purification. In contrast, the GST fusion system could increase protein expression level and the corresponding fusion protein could be easily cleaved by thrombin. After binding to glutathione sepharose, the fusion protein was cleaved on column, and a roughly purified protein fraction was eluted. This fraction was purified by heating at 80 °C for 10 min, followed by centrifugation. The correct total mass and N-terminal primary structure were confirmed by mass spectrometry and Edman degradation. Both constructs were used for in vitro expression, and similar results were obtained, indicating higher expression levels of the GST tag vs. intein/chitin tag. Taken together, our results suggest that the GST fusion system can be used as a considerable alternative to synthetic genes for the expression of rare codon genes. The affinity chromatography purification followed by a heating step is an efficient and convenient method for thermostable protein purification.
Keywords: Rare codon gene;

Yeast protein Yol066 (encoded by YOL066 ORF, also known as Rib2) possesses two distinct sequence domains: C-terminal deaminase domain and N-terminal part related to RNA:pseudouridine (ψ)-synthases. The deaminase domain is implicated in the riboflavine biosynthesis, while the exact function of the RNA:Ψ-synthase domain remains obscure. Here we report the optimisation of growth conditions and purification scheme for recombinant His6-tagged Yol066 expressed in E. coli BL21(DE3) using pET28 plasmid. Production of soluble Yol066 protein is best at low temperature (18 °C) and IPTG concentration (50 μM) and Yol066 purification was achieved using metal-affinity and ion-exchange chromatography. This optimised protocol yields about 10 mg of highly purified recombinant Yol066 from 3 l of E. coli culture.
Keywords: Recombinant Yol066 protein;

The tripartite AcrA–AcrB–TolC system is the major efflux pump of the nosocomial pathogen Enterobacter aerogenes. AcrA is a trimeric periplasmic lipoprotein anchored in the inner membrane, AcrB is an inner membrane transporter and TolC is a trimeric outer membrane channel. In order to reconstitute the AcrA–AcrB–TolC system of E. aerogenes in artificial membranes, we overexpressed and purified the three proteins. The E. aerogenes acrA, acrB and tolC open reading frames were individually inserted in the expression vector pET24a+, in frame with a sequence coding a C-terminal hexahistidine tag to allow purification by INAC (Immobilized Nickel Affinity Chromatography). The mature AcrA–6His was overproduced in a soluble form in the cytoplasm of Escherichia coli BL21(DE3). AcrA–6His was purified under native conditions in two steps using INAC and gel permeation chromatography. We obtained about 25 mg of 97% pure AcrA–6His per liter of culture. AcrB–6His was solubilized from the membrane fraction of E. coli C43(DE3) in 300 mM NaCl, 5% Triton X-100 and purified in one step by INAC. The AcrB–6His enriched fraction was eluted with 100 mM imidazole. The final yield was 1–2 mg of 95% pure AcrB–6His per liter of culture. The membrane fraction of E. coli BL21(DE3)pLysS containing TolC–6His was first treated with 2% Triton X-100, 30 mM MgCl2 to solubilize the inner membrane proteins. After ultracentrifugation, the pellet was treated with 5% Triton X-100, 5 mM EDTA to solubilize the outer membrane proteins. Approximately 5 mg of 95% pure TolC–6His trimers per liter of culture was purified by INAC.

Production of recombinant thermostable proteins expressed in Escherichia coli: completion of protein synthesis is the bottleneck by Hans Peter Sørensen; Hans Uffe Sperling-Petersen; Kim Kusk Mortensen (207-214).
Heterologous expression and high yield purification of proteins are frequently required for structural and functional investigations. Purification of recombinant thermostable proteins is essentially trivial since unwanted mesophilic host protein can efficiently be removed by heat denaturation. However, heterologous expression in E. coli often results in truncated protein forms. In many cases, this is a consequence of abundant codons in heterologous genes, which are decoded by rare tRNAs in E. coli—a combination that can be responsible for translational stalling and termination during protein biosynthesis. Other complications may originate from potential initiation codons and ribosomal binding sites present inside the open reading frame of the target gene or from other less well defined phenomena such as mRNA instability. Separation of full-length protein from truncated forms is a serious chromatographic problem that can be solved in the expression step. We have investigated the heterologous expression and purification of two translation initiation factors from the hyperthermophilic sulphate-reducing archaeon, Archaeoglobus fulgidus. Expression in E. coli was optimised to avoid truncated forms completely by complementation with the plasmids pSJS1244, pRIG, pCODON+ and pLysSR.A.R.E harbouring and expressing genes encoding rare tRNAs corresponding to the codons AGA, AGG, AUA, CUA, GGA, AAG and CCC. Two expression strains, C41(DE3) and C43(DE3) were found highly advantageous when combined with rare tRNA encoding plasmids as compared to BL21(DE3). We have also investigated the effects of site directed mutagenesis on rare lysine encoding AAG doublets as well as two methionine residues preceded by potential ribosomal binding sites. The expression approach presented here has enabled us to purify gram quantities of full-length protein by one step of ion-exchange chromatography and is generally applicable to many other heterologously expressed thermostable proteins.
Keywords: Recombinant thermostable proteins;

Wheat technological properties are correlated with the size of glutenin polymers, consisting of high and low molecular mass glutenin subunits, linked together by disulphide bonds. In order to unravel glutenin polymer structure, we considered three LMW-GS genes, which differ in the number of cysteine residues and in the repetitive domain length. The three LMW-GS genes have been expressed in Escherichia coli, and purified with a yield of 40–100 mg/l of culture volume, depending on protein type. Single polypeptides are being used in re-oxidation and micro-mixographic experiments, in order to detect the influence of the differential structural characteristics on glutenin polymer formation.
Keywords: Low molecular mass glutenin subunits;

Cloning and expression of fatty acids biosynthesis key enzymes from sunflower (Helianthus annuus L.) in Escherichia coli by Marı́a Josefa Serrano-Vega; Mónica Venegas-Calerón; Rafael Garcés; Enrique Martı́nez-Force (221-228).
To further characterize the stearoyl-acyl carrier protein (ACP) desaturase (EC and the acyl-ACP thioesterase FatB (EC activities from sunflower seeds, we cloned, sequenced and expressed the recombinant genes in Escherichia coli. We obtained two partially purified proteins, His-SAD and His-FATB, each of about 45 000 Da. The expression of either proteins produced changes in the E. coli fatty acid profile indicating the functionality of the recombinant proteins. While the expression of His-SAD produced an effect similar to that produced by overexpression of the fabA gene, responsible for the fatty acid desaturation in E. coli, the expression of His-FATB gave rise to an unbalance between unsaturated fatty acids and a toxic effect in E. coli.
Keywords: Fatty acids; Stearoyl-acyl carrier protein desaturase;

Optimisation of expression and immobilized metal ion affinity chromatographic purification of recombinant (His)6-tagged cytochrome P450 hydroperoxide lyase in Escherichia coli by Jérôme Delcarte; Marie-Laure Fauconnier; Philippe Jacques; Kenji Matsui; Philippe Thonart; Michel Marlier (229-236).
Fatty acid hydroperoxide lyase (HPL) is a cytochrome P450 acting on fatty acid’s hydroperoxides in many plants. The optimisation of the expression of recombinant (His)6-tagged HPL in Escherichia coli is described: the highest HPL production yield were obtained with TB medium supplemented with 2.5 mM δ-aminolevulinic acid and 0.5 mM IPTG. For the first time, the time course expression of a plant P450 in a bench-scale fermentor is detailed and the amount of recombinant HPL production is 16.3 mg/l. The UV–Visible spectrum of the recombinant (His)6-tagged HPL have been recorded after a Ni2+-based affinity chromatography (IMAC).
Keywords: Hydroperoxide lyase; Cytochrome P450; Histidine tag;

Continuous processing of fusion protein expressed as an Escherichia coli inclusion body by Giacomo Morreale; Heikki Lanckriet; Jemma C Miller; Anton P.J Middelberg (237-246).
In this study we develop the components of an integrated process for the continuous extraction and purification of a histidine-tagged fusion protein expressed as an inclusion body in Escherichia coli. Lac21 was selected as a model peptide and was expressed as a fusion to ketosteroid isomerase. A purification strategy was developed on a 1-ml batch column before successful scale-up and transfer to a continuous purification system, having a bed volume of 240 ml. Preliminary experiments proved cleavage of the fusion protein. The use of chemical extraction and continuous chromatography gives a flowsheet far superior to the traditional methods for inclusion body processing.
Keywords: Fusion protein;

We recently found that the larger parts of the endocytic proteins epsin 1 and AP180 consist of an unstructured polypeptide chain. As a result these segments are completely heat-stable without loss of their functional properties. We have taken advantage of this fact and developed a combined heat lysis and pre-purification procedure after expressing the disordered domains in E. coli. This results in the irreversible denaturation and precipitation of the majority of bacterial proteins. The bacteria are resuspended in a non-denaturing buffer, heated in a boiling water bath and shock-cooled. We demonstrate that this procedure compared to conventional lysis improves both yield and quality of the purified protein.
Keywords: Intrinsically disordered proteins; Epsin 1; AP180;

Taenia solium cDNA sequence encoding a putative immunodiagnostic antigen for human cysticercosis by E. Montero; L.M. González; L.J.S. Harrison; R.M.E. Parkhouse; T. Gárate (255-269).
A T. solium metacestode cDNA library was prepared and antibody screened to obtain recombinant antigens, which could be used for the neurocysticercosis diagnosis. The F18 clone was selected and sequenced, and the full length cDNA characterised as well as the genomic structure from the gene. F18 is a single copy gene that spans ∼6.1 kb and contains five exons and four introns. The F18 cDNA has a 690-nucleotide open reading frame that encodes a putative polypeptide of 229 amino acids with a predicted molecular mass of 26.06×103 M r. The F18 recombinant protein was obtained and purified by affinity chromatography using pGEX system (G-F18) and pQE system (H-F18). The purified G-F18 fusion protein showed the best results when it was used in ELISA with sera from neurocysticercosis patients.
Keywords: Recombinant proteins; cDNA;

Large scale protein production of the extracellular domain of the transforming growth factor-β type II receptor using the Pichia pastoris expression system by Alwin Scharstuhl; Harrie Glansbeek; Elly L. Vitters; Peter M. van der Kraan; Wim B. van den Berg (271-277).
To study the (patho)physiological role of transforming growth factor-β (TGF-β), potent and selective inhibitors are necessary. Since TGF-β signaling is initiated by the high affinity binding to the type II receptor (RII), the extracellular part of RII (solRII) can function as a TGF-β antagonist. SolRII was cloned and large-scale protein synthesis was performed in the yeast Pichia pastoris expression system. Our results indicate that via this system, high levels of pure concentrated solRII can be obtained. Moreover, purified solRII is an active protein as shown by ELISA and bioassay. In conclusion, our large-scale protein expression procedure results in high quantities of purified solRII, which is a powerful tool to study the natural role of TGF-β.
Keywords: Transforming growth factor β;

Isolation of Qβ polymerase complexes containing mutant species of elongation factor Tu by Sander G.J. Mathu; Charlotte R. Knudsen; Jan van Duin; Barend Kraal (279-286).
The RNA genome of coliphage Qβ is replicated by a complex of four proteins, one of them being the translation elongation factor Tu. The role of EF-Tu in this RNA polymerase complex is still unclear, but the obligate presence of translationally functional EF-Tu in the cell hampers the use of conventional mutational analysis. Therefore, we designed a system based on affinity chromatography and could separate two types of complexes by placing an affinity tag on mutated EF-Tu species. Thus, we were able to show a direct link between the vital tRNA binding property of EF-Tu and polymerase activity.
Keywords: Bacteriophage Qβ; RNA polymerase; Elongation factor-Tu;

The three subunits of the human cardiac troponin complex (hcTnC, hcTnI, hcTnT) were overexpressed in E. coli, purified and reconstituted to form the hcTn complex. This complex was then incorporated into subcellular bundles of mouse cardiac myofibrils whereby the native mcTn complex was replaced. On thus exchanged myofibrils, isometric force kinetics following sudden changes in free Ca2+ concentration were measured using atomic force cantilevers. Following the exchange, the myofibrillar force remained fully Ca2+ regulated, i.e. myofibrils were completely relaxed at pCa 7.5 and developed the same maximum Ca2+-activated isometric force upon increasing the pCa to 4.5 as unexchanged myofibrils. The replacement of endogenous mcTn by wild-type hcTn neither altered the kinetics of Ca2+-induced force development of the mouse myofibrils nor the kinetics of force relaxation induced by the sudden, complete removal of Ca2+. Preparations of functional Tn reconstituted myofibrils provide a promising model to study the role of Tn in kinetic mechanisms of cardiac myofibrillar contraction and relaxation.
Keywords: Troponin; Calcium-regulatory proteins;

Although the functions and antigen recognition requirements of αβ T cells are well characterised, the antigens recognised by γδ T cells and the consequences of this recognition are unclear. γδ T cells are enriched within epithelia, where they eradicate transformed epithelial cells and regulate inflammation. To understand how this occurs, we need to understand the cellular ligands recognised by the γδ cell through the γδ T-cell receptor (TCR). We have therefore generated a soluble TCR (sTCR) to identify ligands for the murine γδ intestinal intraepithelial lymphocyte (IEL) population. sTCR was produced in the baculovirus expression system and purified by affinity chromatography on an anti-TCRδ affinity column. sTCR was recognised by a panel of conformation-specific anti-TCRγδ antibodies. We will now use our sTCR to directly test the binding of putative ligands to the TCR using surface plasmon resonance, and to isolate the ligand biochemically.
Keywords: T-cell receptor;

An exopolyphosphatase gene has been cloned by polymerase chain reaction (PCR) from Trypanosoma brucei and the corresponding protein overexpressed as a recombinant His-tag (histidine tag) exopolyphosphatase in E. coli in order to characterize its biochemical activity and to produce antibody to determine its localization. Because overexpression of this protein in bacteria resulted in the formation of inactive inclusion bodies, these structures were first solubilized in denaturant condition (6 M urea). Secondly, after a capture step using immobilized metal affinity chromatography (IMAC), a gradual refolding of the protein was performed on-column from 6 M to 0 M urea in the presence of 1% Triton X-100. Triton X-100 was used to abolish protein aggregation during the refolding step. The purified enzyme was active, demonstrating that at least part of the proteins was properly refolded.
Keywords: Histidine tag recombinant exopolyphosphatase;

Identification of a tubulin binding motif on the P2X2 receptor by Sandra Gendreau; Jörg Schirmer; Günther Schmalzing (311-318).
To isolate proteins interacting with P2X receptors, GST fusion proteins containing the intracellular C terminal tail of P2X2, P2X5, or P2X7 were used as bait to screen detergent extracts of rat brain synaptosomes. By SDS–PAGE combined with mass spectrometry, two interacting proteins were identified: βIII tubulin and myelin basic protein. While myelin basic protein bound to all three P2X subunits, βIII tubulin interacted exclusively with the P2X2 subunit. The tubulin binding domain could be confined to a proline-rich segment (amino acids 371–412) of the P2X2 subunit. Our results suggest a role for microtubules in the cellular localisation of the P2X2 receptor.
Keywords: β-Tubulin; GST fusion protein; Myelin basic protein; P2X receptor isoforms;

GST–Fbe can recognize β-chains of fibrin(ogen) on explanted materials by Lei Pei; Ingegerd Löfving Arvholm; Lena Lonnies; Jan-Ingmar Flock (319-325).
Staphylococcus epidermidis, a coagulase-negative staphylococcus (CoNS), is one of the leading pathogens of nosocomial infections, particularly associated with foreign body infections. Adherence of S. epidermidis to fibrinogen deposited on the surfaces of implants is important for the development of foreign body infections. A gene (fbe) encoding a fibrinogen-binding protein from S. epidermidis (Fbe) was identified by shotgun phage display. A portion of fbe was cloned into a GST-fusion vector. Affinity to glutathione–Sepharose by the GST-tag and affinity to fibrinogen–Sepharose by the Fbe part were applied to purify the recombinant Fbe. The purity and efficacy of the methods used in protein purification was compared. Furthermore, the potential physiological role of Fbe was studied by the interaction between GST–Fbe and components extracted from explanted materials in vitro.
Keywords: Fibrin; Fibrinogen;

We have generated a single chain antigen binding protein (scFv) recognising morphine. Variable regions of heavy (V H) and light (V L) chain antibody genes isolated from a murine immune repertoire were connected via a glycine–serine linker and cloned into the expression vector pAK 400. The scFv was produced in Escherichia coli JM83 yielding a functional protein of approximately M r 30 000. Immunoaffinity chromatography using M3G–BSA–Sepharose column proved most effective for scFv purification. Purity was monitored by SDS–PAGE and Western blotting and the scFv characterised using ELISA and BIAcore™. The scFv was capable of specifically binding free morphine in solution and was applicable to real sample analysis in saliva. In order to express a bivalent “minibody” the scFv gene was recloned into a vector containing a gene encoding a helix for dimerisation. The scFv was expressed as a protein of M r 75 000 and retained its antibody binding capabilities. Cloning the scFv gene into a vector containing the bacterial alkaline phosphatase gene produced a bifunctional molecule, which retained the binding activity of the parental scFv along with the enzymatic activity of alkaline phosphatase.
Keywords: scFv;

Semi automated production of a set of different recombinant GST-Streptag fusion proteins by Petra Sebastian; Jelena Wallwitz; Stefan Schmidt (343-355).
We describe a high-throughput procedure for the large-scale production of recombinant GST-Streptag fusion proteins. This three-step process, comprising cloning, expression and purification, simultaneously produces up to 96 different proteins in a multi-well format with high yield and purity. Two complementary oligonucleotides, together encoding a specific peptide sequence are annealed and directly ligated into a pre-digested pGEX-2T plasmid carrying an N-terminal GST-tag and a C-terminal Streptag. Following expression, a multichannel pipetting robot purifies the resulting fusion proteins within 2 h by affinity chromatography on Streptactin Macroprep mini-columns.
Keywords: Streptag; Fusion proteins;