Immobilized Metal Chelate Affinity Chromatography Research Articles

Novel endolysin LysMP for control of Limosilactobacillus fermentum contamination in small-scale corn mash fermentation

BackgroundTraditional bioethanol fermentation industries are not operated under strict sterile conditions and are prone to microbial contamination. Lactic acid bacteria (LAB) are often pervasive in fermentation tanks, competing for nutrients and producing inhibitory acids that have a negative impact on ethanol-producing yeast, resulting in decreased yields and stuck fermentations. Antibiotics are frequently used to combat contamination, but antibiotic stewardship has resulted in a shift to alternative antimicrobials.ResultsWe demonstrate that endolysin LysMP, a bacteriophage-encoded peptidoglycan hydrolase, is an effective method for controlling growth of LAB. The LysMP gene was synthesized based on the prophage sequence in the genome of Limosilactobacillus fermentum KGL7. Analysis of the recombinant enzyme expressed in E. coli and purified by immobilized metal chelate affinity chromatography (IMAC) showed an optimal lysis activity against various LAB species at pH 6, with stability from pH 4 to 8 and from 20 to 40 °C up to 48 h. Moreover, it retains more than 80% of its activity at 10% ethanol (v/v) for up to 48 h. When LysMP was added at 250 µg/mL to yeast corn mash fermentations containing L. fermentum, it reduced bacterial load by at least 4-log fold compared to the untreated controls and prevented stuck fermentation. In comparison, untreated controls with contamination increased from an initial bacterial load of 1.50 × 107 CFU/mL to 2.25 × 109 CFU/mL and 1.89 × 109 CFU/mL after 24 h and 48 h, respectively. Glucose in the treated samples was fully utilized, while untreated controls with contamination had more than 4% (w/v) remaining at 48 h. Furthermore, there was at least a fivefold reduction in lactic acid (0.085 M untreated contamination controls compared to 0.016 M treated), and a fourfold reduction in acetic acid (0.027 M untreated contamination controls vs. 0.007 M treated), when LysMP was used to treat contaminated corn mash fermentations. Most importantly, final ethanol yields increased from 6.3% (w/v) in untreated contamination samples to 9.3% (w/v) in treated contamination samples, an approximate 50% increase to levels comparable to uncontaminated controls 9.3% (w/v).ConclusionLysMP could be a good alternative to replace antibiotics for mitigation of LAB contamination in biofuel refineries.

Recombinant production of antibody antigen-binding fragments with an N-terminal human growth hormone tag in mammalian cells

Antigen-binding fragments (Fabs) of antibodies are both key biopharmaceuticals and valuable tools for basic life science. To streamline the production of diverse Fabs by capitalizing on standard and highly optimized protein production protocols, we here explore a method to prepare recombinant Fabs as secreted fusion proteins with an N-terminal human growth hormone domain and an octa-histidine tag. These tagged Fabs can be purified with standard immobilized metal chelate affinity chromatography. We first demonstrated Fab overproduction using the rat monoclonal antibody NZ-1. Optimization of linker residues enabled the complete removal of the tags by TEV protease, leaving only two additional residues at the N-terminus of the heavy chain. We purified NZ-1 Fab at ∼4 μg/mL of culture supernatant and further confirmed that the NZ-1 Fab from the fusion protein maintained its native fold and binding affinity for target cell-surface antigens. We also showed that several other Fabs of mouse IgG1s, the major subclass in mice, could be produced with the same procedure. Our preparation method can provide greater flexibility in functional and structural modifications of target Fabs because specialized purification techniques are not necessary.

Chitosan Co-polymeric nanostructures for catalase immobilization

Catalase is an industrial enzyme used in different areas including medical, textile, food and pharmaceutic industries. In the study, synthetic-natural copolymer in nanosize was synthesized using 2-hydroxyethyl methacrylate (HEMA) as a synthetic material and chitosan (CTS) as a natural material. Immobilized metal-chelate affinity chromatography (IMAC) material was prepared by chelating Cu(II) ions onto p(HEMA-CTS) nanostructures. Characterization studies such as Zeta sizer analysis, Fourier transform infra-red (FTIR) spectroscopy, scanning electron microscopy (SEM), and thermal gravimetric analysis (TGA) were performed during the preparation of IMAC nanostructures. The effects of some parameters (time, polymer amount, pH, initial catalase concentration, ionic strength and temperature) were identified and optimum immobilization conditions were determined as 75 min, 0.1 mg p(HEMA-CTS)-Cu, pH 6.0, and 25 °C. Maximum amount of immobilized catalase was detected as 11.29 g/g p(HEMA-CTS)-Cu. Immobilized catalase was desorbed successfully and it was detected that p(HEMA-CTS)-Cu nanostructures can be used repeatedly in catalase immobilization. Immobilization changed optimum temperature value from 37 °C to 25 °C while it did not change the optimum pH for catalase activity. Furthermore, it was detected that kinetic parameters of catalase changed by immobilization. Finally, operational and storage stabilities of immobilized catalase were investigated.

Substrate and Cofactor Range Differences of Two Cysteine Dioxygenases from Ralstonia eutropha H16.

Cysteine dioxygenases (Cdos), which catalyze the sulfoxidation of cysteine to cysteine sulfinic acid (CSA), have been extensively studied in eukaryotes because of their roles in several diseases. In contrast, only a few prokaryotic enzymes of this type have been investigated. In Ralstonia eutropha H16, two Cdo homologues (CdoA and CdoB) have been identified previously. In vivo studies showed that Escherichia coli cells expressing CdoA could convert 3-mercaptopropionate (3MP) to 3-sulfinopropionate (3SP), whereas no 3SP could be detected in cells expressing CdoB. The objective of this study was to confirm these findings and to study both enzymes in detail by performing an in vitro characterization. The proteins were heterologously expressed and purified to apparent homogeneity by immobilized metal chelate affinity chromatography (IMAC). Subsequent analysis of the enzyme activities revealed striking differences with regard to their substrate ranges and their specificities for the transition metal cofactor, e.g., CdoA catalyzed the sulfoxidation of 3MP to a 3-fold-greater extent than the sulfoxidation of cysteine, whereas CdoB converted only cysteine. Moreover, the dependency of the activities of the Cdos from R. eutropha H16 on the metal cofactor in the active center could be demonstrated. The importance of CdoA for the metabolism of the sulfur compounds 3,3'-thiodipropionic acid (TDP) and 3,3'-dithiodipropionic acid (DTDP) by further converting their degradation product, 3MP, was confirmed. Since 3MP can also function as a precursor for polythioester (PTE) synthesis in R. eutropha H16, deletion of cdoA might enable increased synthesis of PTEs.

Immobilized metal ion affinity nanospheres for $\alpha $-amylase immobilization

Immobilized metal chelate affinity chromatography (IMAC) support was practiced for a-amylase immobilization. Poly(hydroxyethylmethacrylate-methacryloylamidotryptophan)-Ni2+ [p(HEMA-MAT)-Ni2+] nanospheres, average diameter 100 nm, were produced by surfactant free emulsion polymerization. Characterizations of p(HEMA-MAT)-Ni2+ nanospheres were carried out by Fourier transform infrared (FTIR) spectroscopy and scanning electron microscope (SEM). In addition, average particle size, size distribution, and surface charge were specified. The amount of N-methacryloylamidotryptophan (MAT) incorporated to polymer was determined as 1.95 mmol/g polymers by using nitrogen stoichiometry. The specific surface areas of poly(hydroxyethylmethacrylate) [p(HEMA)] and p(HEMA-MAT) nanospheres were calculated as 1856 m2/g and 1914 m2/g, respectively. Protein adsorption increased with increasing initial protein concentration and maximum a-amylase adsorption on p(HEMA-MAT)-Ni2+ nanospheres was observed at pH 4.0. Both free and immobilized a-amylase showed pH optimum at pH 7.0. It was determined that the immobilized a-amylase had better thermostability than the free one. Immobilization of the enzyme did not significantly change the kinetic parameters. The storage stability of a-amylase increased upon immobilization. It was also observed that p(HEMA-MAT)-Ni2+ nanospheres can be repeatedly used for a-amylase immobilization.

Abstract B32: Cell penetrating Myosin-Va fragment covering the binding motif of DLC2: Evaluation of pro-apoptotic properties

Abstract Previous studies proposed that myosin-Va regulates apoptosis by sequestering pro-apoptotic Bmf to the actin cytoskeleton through dynein light chain-2 (DLC2). Adhesion loss or other cytoskeletal perturbations would unleash Bmf, allowing it to bind and inhibit pro-survival Bcl2 proteins. Recently, our research group has demonstrated that overexpression of a myosin-Va medial tail fragment (MVaf) harboring the binding site for DLC2 dramatically decreased melanoma cell viability. Morphological and molecular changes, including surface blebbing, mitochondrial outer membrane permeabilization, cytochrome-c and Smac release, as well as caspase-9/-3 activation and DNA fragmentation indicated that melanoma cells died of apoptosis. Besides, MVaf expression attenuated B16-F10 solid tumor growth in mice suggesting that this peptide may be useful as an apoptosis-inducing tool for basic cancer research. We suggest that MVaf induces apoptosis by sequestering DLC2 and DLC1, thereby unleashing the pair of sensitizer and activator BH3-only proteins Bmf and Bim. In view of such findings, we have proposed to develop a recombinant Tat-fusion MVaf to be explored in peptide-based therapeutic approaches. The native MVaf sequence was altered through the N-terminal addition of a transducible Tat-HA tag. EGFP was alternatively introduced into recombinant products to assess their subcellular localization. Bacterially expressed His-tagged fusion proteins were purified by immobilized metal chelate affinity chromatography on Ni-NTA beads, and transduction assays were initially performed in HeLa cells. The kinetics of Tat-HA-MVaf (THM, 13.7 kDa) and Tat-HA-EGFP-MVaf (THEM, 41.9 kDa) uptake, at concentrations ranging from 1 to 2 μM, was evaluated by confocal laser scanning microscopy (CLSM). Tat-HA-EGFP (THE, 33.9 kDa) was used as a control. Tat-HA-MVaf displayed a diffuse distribution throughout the cells in the first 5 min incubation, with a clear perinuclear accumulation after 1h incubation. THEM and THE fluorescence were detected in many developing vesicles adjacent to cell membrane, and distributed throughout the cytoplasm and nucleus after 5 min incubation, but became markedly weaker after 1h, suggesting proteolytic loss. Despite intense diffusion and intracellular accumulation of recombinant proteins, no significant changes in morphology and cell viability were detected by means of flow cytometry and MTT assay. Pull-down assay revealed that THEM but not THE binds to immobilized GST-DLC2 and to GST-DLC1 in a lesser extent, which confirms peptide-binding capacity to DLCs. On the other hand, pretreatment of cells with chloroquine, an inhibitor of endosomal acidification, sensitized cells to peptide treatment. We believe that Tat-mediated subcellular localization may have predominated, resulting in mislocalization of the peptide cargo. Also, one major drawback in using pTAT-conjugated peptides is their entrapment in cytoplasmic compartments and nucleus, resulting in limited availability and biological activity. One possible strategy is to produce recombinant peptides conjugated reversibly to Tat by a disulfide bond, which are disrupted upon intracellular delivery. Funding support. FAPESP / CNPq. Citation Format: Denise P. S. Leitão Mazzi, Cleidson P. Alves, Enilza Maria Espreafico. Cell penetrating Myosin-Va fragment covering the binding motif of DLC2: Evaluation of pro-apoptotic properties. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B32.

Molecular characterization and enzymatic hydrolysis of naringin extracted from kinnow peel waste

Kinnow peel, a waste rich in glycosylated phenolic substances, is the principal by-product of the citrus fruit processing industry and its disposal is becoming a major problem. This peel is rich in naringin and may be used for rhamnose production by utilizing α- l-rhamnosidase (EC 3.2.1.40), an enzyme that catalyzes the cleavage of terminal rhamnosyl groups from naringin to yield prunin and rhamnose. In this work, infrared (IR) spectroscopy confirmed molecular characteristics of naringin extracted from kinnow peel waste. Further, recombinant α- l-rhamnosidase purified from Escherichia coli cells using immobilized metal-chelate affinity chromatography (IMAC) was used for naringin hydrolysis. The purified enzyme was inhibited by Hg 2+ (1 mM), 4-hydroxymercuribenzoate (0.1 mM) and cyanamide (0.1 mM). The purified enzyme established hydrolysis of naringin extracted from kinnow peel and thus endorses its industrial applicability for producing rhamnose.

Molecular Cloning and Expression of Bacterial Mercuric Reductase Gene

In order to characterize the bacterial mercuric reductase (merA) gene, mercury resistant (Hgr) Escherichia coli strains have been isolated from various mercury contaminated sites of India. Their minimum inhibitory concentration (MIC) for Hg and zone of inhibition for different antibiotics were measured, and finally mer operon was localized by transforming isolated E. coli plasmid into mercury sensitive (Hgs) host E. coli DH5a cells. Oligonucleotide primers were designed by comparing the known reported sequences of merA from Gram-negative bacterium (E. coli plasmid R100) and 1695 bp full length merA gene was amplified by PCR. A 1.695-kb DNA fragment of merA was inserted into isopropyl-
-D-thiogalactopyranoside (IPTG) inducible bacterial expression vector pQE-30U/A. E. coli DH5 strains harboring the merA constructs showed higher mercury reductase enzyme (MerA) activity and expressed significantly more MerA than the control strains under aerobic conditions. The purified merA gene over expressed in the specific host E. coli BL21(DE3)Plys cells. Finally, expressed MerA protein was purified by Immobilized Metal-chelate Affinity Chromatography (IMAC) by using Ni- NTA column; and ~66.2 kDa bacterial MerA protein was detected after resolving on 10% sodium dodecyl sulphate poly acrylamide gel electrophoresis (SDS PAGE).

One-step purification and immobilization of His-tagged rhamnosidase for naringin hydrolysis

α- l-Rhamnosidase (EC 3.2.1.40) is an enzyme that catalyzes the cleavage of terminal rhamnoside groups from naringin to prunin and rhamnose. In this study, a His-tag was genetically attached to the rhamnosidase gene ramA from Clostridium stercorarium to facilitate its purification from Escherichia coli BL21 (DE3) cells containing the pET-21d/ramA plasmid. Immobilized metal-chelate affinity chromatography (IMAC) resulted in one-step purification of N-terminally His-tagged recombinant rhamnosidase (N-His-CsRamA) which was immobilized in Ca 2+ alginate (3%) beads. The optimum pH levels of the free and immobilized recombinant rhamnosidase were found to be 6.0 and 7.5, and the optimum temperature 55 and 60 °C respectively. At 50 °C, the free enzyme was relatively stable and exhibited a less than 50% reduction in residual activity after 180 min of incubation. The free and immobilized enzymes achieved 76% and 67% hydrolysis of the naringin in Kinnow juice respectively. Immobilization of recombinant rhamnosidase enabled its reutilization up to 9 hydrolysis batches without an appreciable loss in activity. This result indicated that the His-tagged thermostable rhamnosidase could be prepared as described and may serve to illustrate an economical and commercially viable process for industrial application.

Characterization of a Rab11 homologue, EoRab11a, inEuplotes octocarinatus

Rab GTPases are crucial in the regulation of intracellular vesicular trafficking. A novel Rab GTPase gene, EoRab11a (GenBank accession no. EF061065), was isolated and identified from Euplotes octocarinatus cells in this study. It contains an ORF of 696-bp nucleotides, encoding 231 amino acids with a calculated molecular weight of 26.8 kDa. Alignment of EoRab11a with other Rab11 proteins from other eukaryotes demonstrated that these proteins shared 53-61% identity at the amino acid level. The recombinant EoRab11a was expressed in Escherichia coli and purified by immobilized metal chelate affinity chromatography and iron chromatography. The GTPase activity of EoRab11a was 0.0024 min(-1) detected by HPLC at 30 degrees C. Three mutations were generated at amino acids Ser21 and Gly22 positions in the G1 domain of EoRab11a. All three mutants, S21P, S21G and G22R, increased the GTPase activity in vitro. Immunofluorescence microscopy results indicated that EoRab11a was localized on the phagosomal membrane during phagocytosis of E. octocarinatus. These data show that EoRab11a possesses GTP hydrolysis activity and may participate in vesicle transport events during phagocytosis of E. octocarinatus.

3-Mercaptopropionate Dioxygenase, a Cysteine Dioxygenase Homologue, Catalyzes the Initial Step of 3-Mercaptopropionate Catabolism in the 3,3-Thiodipropionic Acid-degrading Bacterium Variovorax paradoxus

The thioether 3,3-thiodipropionic acid can be used as precursor substrate for biotechnological synthesis of 3-mercaptopropionic acid-containing polythioesters. Therefore, the hitherto unknown catabolism of this compound was elucidated to engineer novel and improved polythioester biosynthesis pathways in the future. Bacteria capable of using 3,3-thiodipropionic acid as the sole source of carbon and energy for growth were enriched from the environment. From eleven isolates, TBEA3, TBEA6, and SFWT were morphologically and physiologically characterized. Their 16 S rDNAs and other features affiliated these isolates to the beta-subgroup of the proteobacteria. Tn5::mob mutagenesis of isolate Variovorax paradoxus TBEA6 yielded ten mutants fully or partially impaired in growth on 3,3-thiodipropionic acid. Genotypic characterization of two 3,3-thiodipropionic acid-negative mutants demonstrated the involvement of a bacterial cysteine dioxygenase (EC 1.13.11.22) homologue in the further catabolism of the 3,3-thiodipropionic acid cleavage product 3-mercaptopropionic acid. Detection of 3-sulfinopropionate in the supernatant of one of these mutants during cultivation on 3,3-thiodipropionic acid as well as in vivo and in vitro enzyme assays using purified protein demonstrated oxygenation of 3-mercaptopropionic acid to 3-sulfinopropionate by this enzyme; cysteine and cysteamine were not used as substrate. Beside cysteine dioxygenase and cysteamine dioxygenase, this 3-mercaptopropionic acid dioxygenase is the third example for a thiol dioxygenase and the first report about the microbial catabolism of 3-mercaptopropionic acid. Insertion of Tn5::mob in a gene putatively coding for a family III acyl-CoA-transferase resulted in the accumulation of 3-sulfinopropionate during cultivation on 3,3-thiodipropionic acid, indicating that this compound is further metabolized to 3-sulfinopropionyl-CoA and subsequently to propionyl-CoA.

One step purification–immobilization of fuculose-1-phosphate aldolase, a class II DHAP dependent aldolase, by using metal-chelate supports

His-tagged recombinant fuculose-1-phosphate aldolase (FucA) from E. coli has been purified by immobilized metal-chelate affinity chromatography (IMAC) at gram scale. During this operation, there was a metal exchange between FucA and the affinity matrix, being the purification yields dependent on the metal nature, which was bound to affinity matrix. One step purification–immobilization of FucA has been carried out on metal-chelate support. The preparation of a FucA immobilized derivative, available to be used as catalyst in aldol addition reactions, has been accomplished in a single step starting from E. coli cell extracts. The best results were obtained with high density support containing Co 2+. The immobilization yield was 100% and the immobilized derivative showed 63% of FucA activity initially offered to the support. The best derivative of immobilized FucA is 21-fold more stable than the soluble FucA in DMF/buffer (1:4) at 25 °C and it catalyzes aldol addition between S-Cbz-Alaninal and DHAP.

Characterization of aquatic humic substances and their metal complexes by immobilized metal-chelate affinity chromatography on iron(III)-loaded ion exchangers.

The analytical fractionation of aquatic humic substances (HS) by means of immobilized metal-chelate affinity chromatography (IMAC) on metal-loaded chelating ion exchangers is described. The cellulose HYPHAN, loaded with different trivalent ions, and the chelate exchanger Chelex 100, loaded to 90% of its capacity with Fe(III), were used. The cellulose HYPHAN, loaded with 2% Fe(III), resulted in HS distribution coefficients Kd of up to 10(3.7) mL/g at pH 4.0 continuously decreasing down to 10(1.5) at pH 12, which were appropriate for HS fractionation by a pH-depending chromatographic procedure. Similar distribution coefficients Kd were obtained for HS sorption onto Fe(III)-loaded Chelex 100. On the basis of Fe-loaded HYPHAN both, a low-pressure and high-pressure IMAC technique, were developed for the fractionation of dissolved HS applying a buffer-based pH gradient for their gradual elution between pH 4.0 and 12.0. By coupling the Chelex 100 column under high-pressure conditions with an inductively coupled plasma mass spectrometer an on-line characterization of HS metal species could be achieved. Using these fractionation procedures a number of reference HS were characterized. Accordingly, the HA (humic acids) and FA (fulvic acids) studied could be discriminated into up to 6 fractions by applying cellulose HYPHAN, significantly differing in their Cu(II) complexation capacity but hardly in their substructures assessed by conventional FTIR. In the case of using Chelex 100 exchanger resin two major UV active HS fractions were obtained, which significantly differ in their complexation properties for Cu(II) and Pb(II), respectively.

Purification of his-tagged proteins by immobilized chelate affinity chromatography: the benefits from the use of organic solvent.

Recombinant proteins overexpressed in and purified from Escherichia coli contain impurities that are toxic in biological assays. The application of affinity purification procedures is often not sufficient to remove these toxic components. We here describe a simple and fast, one-step protocol to remove these impurities highly efficiently. Four recombinant proteins were overexpressed in E. coli as His-tagged fusion proteins and purified by immobilized metal chelate affinity chromatography on Ni-NTA beads. Depending on the protein, the composition of the lysis buffer, and the washing protocol, various impurities appeared to be present in the purified protein preparations. Here we show how the use of 60% isopropanol during washing steps removed most of these contaminants from the end products. In addition to the removal of proteins that aspecifically adhere to the beads or to the tagged protein, this procedure was particularly useful in removing endotoxins. Moreover, we show that detergents such as NP-40, that are necessarily employed during lysis, are also efficiently removed. Finally, we show that proteins are able to refold correctly after isopropanol treatment. Thus, the resulting end products contain significantly less contaminating E. coli proteins, endotoxins, and detergents.

Production of the Islet Cell Antigen ICA69 (p69) with: Baculovirus Expression System: Analysis with a: Solid-phase Time-resolved Fluorescence Method of Sera: from Patients with IDDM and Rheumatoid Arthritis

Islet cell antigen 69 (ICA69), previously implicated as an autoantigen in autoimmune insulin-dependent diabetes mellitus (IDDM), was produced using baculovirus-mediated expression in Spodoptera frugiperda (Sf9) insect cells. In these cells the protein was effectively expressed and ICA69 carrying C-terminal histidine-hexapeptide could be efficiently purified using immobilized metal chelate affinity chromatography. Screening of patient and control sera using this protein as an antigen in time-resolved fluoroimmunoassay (TR-FIA) identified 4/50 of patients with IDDM and 6/73 of patients with rheumatoid arthritis (RA) to be positive for ICA69 antibodies. The number of positives did not differ significantly between patients and control subjects but the level of binding was higher in sera from RA patients compared to that of control sera (P = 0.003). The results show that some subjects have specific autoreactive antibodies against the ICA69 protein produced with recombinant technology.

Encephalitogenicity of myelin-associated oligodendrocytic basic protein and 2',3'-cyclic nucleotide 3'-phosphodiesterase for BALB/c and SJL mice.

In search of new encephalitogenic myelin antigens, the 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) and 19 000 MW isoform of myelin-associated oligodendrocytic basic protein (MOBP) were obtained as recombinant proteins by the baculovirus expression system in Spodoptera frugiperda cells and purified to homogeneity by immobilized metal chelate affinity chromatography (IMAC). The purified MOBP was soluble in water and showed retarded migration on sodium dodecyl sulphate-polyacrylamide gel electrophoresis similar to myelin basic protein (MBP). MOBP induced experimental autoimmune encephalomyelitis (EAE) in nine of 15 susceptible SJL OlaHsd mice, causing death in two animals, whereas three of 14 BALB/c mice showed mild symptoms of EAE, manifested as transient weakness of hind limbs. In both mouse strains, periventricular infiltrates of mononuclear cells were observed. In addition, both 46 000 MW and 48 000 MW CNP isoforms were shown to be non-encephalitogenic for both mouse strains.

Cloning, expression and characterization of a single‐chain antibody specific for the herbicide atrazine

The antigen‐binding domain of an antibody specific for the herbicide atrazine was cloned from hybridoma 4063–21–1 and expressed as a single‐chain antibody (scAb) in the vector pPM1‐His. The observed heavy and light chain gene sequences were consistent with those in subgroup VH I (B) and VK TV, as defined by the Kabat database of sequences. Subsequent expression in Escherichia coli strain XL‐1 Blue produced up to 0.3 μg of functional single‐chain antibody, from periplasmic protein preparations, per millilitre of culture. The scAb was purified as a monomer by immobilized metal chelate affinity chromatography via a hexa‐histidine tail or as a dimer by antibody affinity chromatography. The functionality and specificity of the recombinant antibody was confirmed by binding to an atrazine‐bovine serum albumin conjugate and free atrazine and simazine in a competition ELISA. This is the first example of the production and purification of a recombinant antibody that retains antigen binding for a triazine herbicide.

New approaches to chromatographic purification of bovine dopamine-beta-hydroxylase.

The use of the traditional scheme for the isolation of bovine dopamine-beta-hydroxylase (bDBH) from bovine adrenal medulla resulted in active but not pure bDBH, containing about 50% of admixtures. Immobilized metal chelate affinity chromatography on agarose modified with iminodiacetic acid residues and charged with cobalt ions was applied in the final stage to obtain more than 90% pure and active bDBH. Final purification of bDBH using step elution with 0-0.5 M methyl-D-mannoside in buffer solution from concanavalin A-Sepharose was studied. The determination of bDBH in various samples was performed using size-exclusion chromatography.

Bacterial expression of human chorionic gonadotropin α subunit: studies on refolding, dimer assembly and interaction with two different β subunits

Human chorionic gonadotropin (hCG) is a member of a family of heterodimeric glycoprotein hormones that have a common α subunit but differ in their hormone-specific β subunit. The common α subunit contains two asparagine (N)-linked oligosaccharides. To study the function of carbohydrates on in vitro refolding of α subunit and dimer assembly, we generated recombinant non-glycosylated hCG α subunit (rNG-hCGα) from E. coli. The expression vector was constructed by inserting hCGα cDNA coding for the mature form in-frame into a pQE-30 vector, which contains a 6 × His sequence immediately before the 5′-end of hCGα cDNA for subsequent purification of rNG-hCGα. The rNG-hCGα expressed in inclusion bodies was efficiently purified by immobilized metal chelate affinity chromatography on Ni-NTA resin. SDS-PAGE, solid-phase binding assay and immunoblotting demonstrated the expression of rNG-hCG. Its α molecular weight on SDS-PAGE was 14.7 kDa under reducing conditions and 15 kDa for a monomer accompanied with some higher molecular weight oligomer under non-reducing conditions. Reconstitution of rNG-hCGα with native hCGβ and oFSHβ occurred in very low yield under standard conditions. However, the oxidation-reduction system cystamine (1.34 mM) and cysteamine (7.3 mM) facilitated both the refolding of rNG-hCGα and reconstitution of rNG-hCGα with native hCGβ to regain partially correct conformation. These were revealed by conformationally sensitive antibody and receptor binding assays. Cystamine and cysteamine were more effective in the recombination of rNG-hCGα with oFSHβ as indicated by a 22–36-fold decrease in the amount required to cause a 50% competitive inhibition in radioreceptor assay. They have no effect on assembly of rNG-hCGα with oLHβ. Our results suggest the carbohydrate moieties confer greater conformational flexibility to the backbone of the β subunit and the relative rigidity of the β subunit may serve as a conformational template of the α subunit. The present approach has made it possible to prepare the non-glycosylated gonadotropin α subunit in adequate amounts for further study on their biological and topographical features in complete absence of carbohydrate.

Purification of two active fusion proteins of the Na +-dependent citrate carrier of Klebsiella pneumoniae

The sodium-ion-dependent citrate carrier of Klebsiella pneumoniae (CitS) was purified by means of bioengineerical methods. By fusing the biotin acceptor domain of the α-subunit of the oxaloacetate decarboxylase of K pneumoniae to the C-terminus of CitS, purification of the carrier was achieved by use of a monomeric avidin-Sepharose column. Additionally, we were able to purify a CitS-protein with an N-terminal histidine-tag by immobilized metal chelate affinity chromatography (with Ni 2+-nitrilotriacetic acid-(NTA-) resin). Both purified fusion proteins showed citrate transport activity after reconstitution into liposomes by the freeze/thaw/sonication procedure.