Development of a cell surface display system in a magnetotactic bacterium, "Magnetospirillum magneticum" AMB-1

Bacterial cell surface display is a widely used technology for bioadsorption and for the development of a variety of screening systems. Magnetotactic bacteria are unique species of bacteria due to the presence of magnetic nanoparticles within them. These intracellular, nanosized (50 to 100 nm) magnetic nanoparticles enable the cells to migrate and be manipulated by magnetic force. In this work, using this unique characteristic and based on whole-genomic and comprehensive proteomic analyses of these bacteria, a cell surface display system has been developed by expressing hexahistidine residues within the outer coiled loop of the membrane-specific protein (Msp1) of the "Magnetospirillum magneticum" (proposed name) AMB-1 bacterium. The optimal display site of the hexahistidine residues was successfully identified via secondary structure prediction, immunofluorescence microscopy, and heavy metal binding assay. The established AMB-1 transformant showed high immunofluorescence response, high Cd(2+) binding, and high recovery efficiency in comparison to those of the negative control when manipulated by magnetic force.     

Soluble expression in Escherichia coli,purification and characterization of a human TF-1 cell apoptosis-related protein TFAR19

A novel human TF-1 cell apoptosis-related protein, TFAR19, cloned from a human leukemia cell line, TF-1, was first overexpressed in Escherichia coli with the sequence Met-Gly-His(6)-Gly-Thr-Asn-Gly, a hexahistidine sequence followed by a hydroxylamine cleavage site attached to its amino terminus. The resulting protein was soluble and single-step purified to homogeneity by metal chelating affinity chromatography. After cleavage of the purified His(6)-tagged TFAR19 sample with hydroxylamine, highly purified untagged TFAR19 protein was then obtained through an FPLC Resource Q column. The structural characteristics and function of the His(6)-tagged and untagged TFAR19 proteins were studied using circular dichroism, intrinsic fluorescence, and ANS-binding fluorescence spectra and apoptosis activity assay. The results show that alpha-helix is the main secondary structure of the proteins and the two forms of TFAR19 protein fold properly, which correspond well to their apoptosis activity expression. The results also indicate that the extra sequence including the His(6)-tag fused to the N-terminus of TFAR19 protein has a minimal effect on its structure and function, suggesting that the His(6)-tagged TFAR19 protein could be further used as an immobilized target for finding potential proteins which interact with TFAR19 from a cDNA library using in vitro ribosome display technique.     

Familial amyotrophic lateral sclerosis-associated mutations decrease the thermal stability of distinctly metallated species of human copper/zinc superoxide dismutase

We report the thermal stability of wild type (WT) and 14 different variants of human copper/zinc superoxide dismutase (SOD1) associated with familial amyotrophic lateral sclerosis (FALS). Multiple endothermic unfolding transitions were observed by differential scanning calorimetry for partially metallated SOD1 enzymes isolated from a baculovirus system. We correlated the metal ion contents of SOD1 variants with the occurrence of distinct melting transitions. Altered thermal stability upon reduction of copper with dithionite identified transitions resulting from the unfolding of copper-containing SOD1 species. We demonstrated that copper or zinc binding to a subset of "WT-like" FALS mutants (A4V, L38V, G41S, G72S, D76Y, D90A, G93A, and E133Delta) conferred a similar degree of incremental stabilization as did metal ion binding to WT SOD1. However, these mutants were all destabilized by approximately 1-6 degrees C compared with the corresponding WT SOD1 species. Most of the "metal binding region" FALS mutants (H46R, G85R, D124V, D125H, and S134N) exhibited transitions that probably resulted from unfolding of metal-free species at approximately 4-12 degrees C below the observed melting of the least stable WT species. We conclude that decreased conformational stability shared by all of these mutant SOD1s may contribute to SOD1 toxicity in FALS.     

Metal ions‐binding T4 lysozyme as an intramolecular protein purification tag compatible with X‐ray crystallography

Phage T4 lysozyme is a well folded and highly soluble protein that is widely used as an insertion tag to improve solubility and crystallization properties of poorly behaved recombinant proteins. It has been used in the fusion protein strategy to facilitate crystallization of various proteins including multiple G protein‐coupled receptors, lipid kinases, or sterol binding proteins. Here, we present a structural and biochemical characterization of its novel, metal ions‐binding mutant (mbT4L). We demonstrate that mbT4L can be used as a purification tag in the immobilized‐metal affinity chromatography and that, in many respects, it is superior to the conventional hexahistidine tag. In addition, structural characterization of mbT4L suggests that mbT4L can be used as a purification tag compatible with X‐ray crystallography.     

Separation of hexahistidine fusion proteins with immobilized metal ion affinity chromatographic (IMAC) sorbents derived from MN+‐tacn and its derivatives

The capabilities of a new class of immobilized (im) metal ion chelate complexes (IMCCs), derived from 1,4,7‐triazacyclononane (tacn), bis(1,4,7‐triazacyclononyl) ethane (dtne) and bis(1,4,7‐triazacyclononyl)propane (dtnp) complexed with the borderline metal ions Cu²⁺, Ni²⁺, Zn²⁺, Mn²⁺, Co²⁺, and Cr³⁺, for the purification of proteins have been investigated. In particular, the binding behavior of a model protein, the C‐terminal hexahistidine tagged recombinant fusion protein Schistosoma japonicum glutathione S‐transferase‐Saccharomyces cerevisiae mitochondrial ATP synthase δ‐subunit (GST‐δATPase‐His₆), with these new immobilized metal ion affinity chromatographic (IMAC) sorbents was compared to the properties of a conventional sorbent, derived from immobilized Ni(II)‐nitrilotriacetic acid (im‐Ni²⁺‐NTA). Investigations using the recombinant GST‐δATPase‐His₆ and recombinant S. japonicum glutathione S‐transferase (GST) lacking a hexahistidine tag have confirmed that the C‐terminal tag hexahistidine residues were required for the binding process to occur with these IMAC systems. The results also confirm that recombinant fusion proteins such as GST‐δATPase‐His₆ can be isolated in high purity with these IMAC systems. Moreover, these new macrocyclic systems manifest different selectivity features as a function of pH or ionic strength when compared to the conventional, unconstrained iminodiacetic acid (IDA) or NTA chelating ligands, complexed with borderline metal ions such as Cu²⁺ or Ni²⁺, as IMAC systems. Biotechnol. Bioeng. 2009;103: 747–756. © 2009 Wiley Periodicals, Inc.     

High-level expression of his-tagged clostridial collagenase in Clostridium perfringens

Clostridium histolyticum collagenase is used to isolate cells from various organs and tissues for tissue engineering, and also to treat destructive fibrosis; thus, the demand for high-grade enzyme preparations is increasing. In this study, we constructed a plasmid encoding C. histolyticum type II collagenase (ColH) with a C-terminal hexahistidine tag (ColH-his) to facilitate the purification of the enzyme through immobilized metal affinity chromatography (IMAC). When ColH-his was expressed in a protease-deficient mutant of Clostridium perfringens, it was produced in the culture supernatant more efficiently than the untagged ColH. ColH-his exhibited the same hydrolytic activity as ColH against 4-phenylazobenzyloxy-carbonyl-Pro-Leu-Gly-Pro-D: -Arg (Pz peptide), a synthetic collagenase substrate. From 100 ml of the culture supernatant, approximately 1 mg of ColH-his was purified by ammonium sulfate precipitation, IMAC, and high-performance liquid chromatography on a MonoQ column. When IMAC was performed on chelating Sepharose charged with Zn(2+) instead of Ni(2+), a potential carcinogenic metal, the specific activities against Pz peptide and type I collagen decreased slightly. However, they were comparable to those reported for other recombinant ColHs and a commercial C. histolyticum collagenase preparation, suggesting that this expression system is useful for large-scale preparation of high-grade clostridial collagenases.     

Development of a Bacterial Surface Display of Hexahistidine Peptide Using CS3 Pili for Bioaccumulation of Heavy Metals

A novel cell surface display system for metal uptake was developed using CS3 pili of enterotoxigenic Escherichia coli, which is a suitable system for display of heterologous peptides. The recombinant bacteria producing the hybrid pili containing the hexahistidine peptide accumulated high concentrations of Cd²⁺ and Ni²⁺ at 656.2 and 276.5 nmol per mg dry weight of bacterial cell, respectively. The recombinant bacteria may be useful in water and waste water treatment.     

Expression, purification and spectroscopic studies of full-length Kir3.1 channel C-terminus

A polypeptide corresponding to the full-length C-terminal cytoplasmic domain of a G-protein-regulated inwardly rectifying potassium channel (Kir3.1) bearing a hexahistidine (His6) tag was produced by DNA recombinant overexpression techniques in Escherichia coli. This permitted the isolation of approximately 5 mg of pure protein per liter of bacterial culture. Further purification by size exclusion chromatography (SEC) of the C-terminal domain revealed that it exists predominantly as a dimer. The secondary structure was estimated using circular dichroism measurements that indicated the presence of approximately 35% beta-sheet and approximately 15% alpha-helix. G-protein betagamma subunits incubated with His-tagged Kir3.1 C-terminal domain, bound to immobilized metal affinity chromatography (IMAC) resin, copurified with the peak of specifically eluted recombinant protein. These observations demonstrate that full-length Kir3.1 C-terminus can be purified in a stable conformation capable of binding proteins known to activate Kir3 channels and may contain elements involved in channel assembly.     

Expression of recombinant Atlantic salmon serum C-type lectin in Drosophila melanogaster Schneider 2 cells

The Atlantic salmon (Salmo salar) serum lectin (SSL) is a soluble C-type lectin that binds bacteria, including salmon pathogens. This lectin is a cysteine-rich oligomeric protein. Consequently, a Drosophila melanogaster expression system was evaluated for use in expressing SSL. A cDNA encoding SSL was cloned into a vector designed to express it as a fusion protein with a hexahistidine tag, under the control of the Drosophila methallothionein promoter. The resulting construct was stably transfected into Drosophila S2 cells. After CdCl₂ induction, transfected S2 cells secreted recombinant SSL into the cell culture medium. A cell line derived from stably transformed polyclonal cell populations expressing SSL was used for large-scale expression of SSL. Recombinant SSL was purified from the culture medium using a two-step purification scheme involving affinity binding to yeast cells and metal-affinity chromatography. Although yields of SSL were very low, correct folding and functionality of the recombinant SSL purified in this manner was demonstrated by its ability to bind to Aeromonas salmonicida. Therefore, Drosophila S2 cells may be an ideal system for the production of SSL if yields can be increased.     

A surface plasmon resonance-based assay for small molecule inhibitors of human cyclophilin A

A simple protocol for generating a highly stable and active surface plasmon resonance (SPR) sensor surface of recombinant human hexahistidine cyclophilin A (His-CypA) is described. The sensor surface was sensitive and stable enough to allow, for the first time, the screening and ranking of several novel small-molecule (Mr approximately 250-500 Da) ligands in a competition binding assay with cyclosporin A (CsA). It also allowed us to accurately determine the kinetic rate constants for the interaction between His-CypA and CsA. His-CypA was first captured on a Ni2+-nitrilotriacetic acid (NTA) sensor chip and was then briefly covalently stabilized, coupling via primary amines. The significant baseline drift observed due to dissociation of weakly bound His-CypA from the Ni2+-NTA moiety was eliminated, resulting in a surface that was stable for at least 36 h. In addition, immobilized protein activity levels were high, typically between 85 and 95%, assayed by the interaction between His-CypA and CsA. The mean equilibrium dissociation constant for CsA (K(dCsA)) binding to the immobilized His-CypA was 23+/-6 nM, with on and off rates of 0.53+/-0.1 microM(-1) s(-1) and 1.2+/-0.1 (x 10(-2)) s(-1), respectively. These values agree well with the values for the corresponding binding constants determined from steady-state and kinetic fluorescence titrations in solution.     

Inositol phosphates promote HIV-1 assembly and maturation to facilitate viral spread in human CD4+ T cells

Gag polymerization with viral RNA at the plasma membrane initiates HIV-1 assembly. Assembly processes are inefficient in vitro but are stimulated by inositol (1,3,4,5,6) pentakisphosphate (IP5) and inositol hexakisphosphate (IP6) metabolites. Previous studies have shown that depletion of these inositol phosphate species from HEK293T cells reduced HIV-1 particle production but did not alter the infectivity of the resulting progeny virions. Moreover, HIV-1 substitutions bearing Gag/CA mutations ablating IP6 binding are noninfectious with destabilized viral cores. In this study, we analyzed the effects of cellular depletion of IP5 and IP6 on HIV-1 replication in T cells in which we disrupted the genes encoding the kinases required for IP6 generation, IP5 2-kinase (IPPK) and Inositol Polyphosphate Multikinase (IPMK). Knockout (KO) of IPPK from CEM and MT-4 cells depleted cellular IP6 in both T cell lines, and IPMK disruption reduced the levels of both IP5 and IP6. In the KO lines, HIV-1 spread was delayed relative to parental wild-type (WT) cells and was rescued by complementation. Virus release was decreased in all IPPK or IPMK KO lines relative to WT cells. Infected IPMK KO cells exhibited elevated levels of intracellular Gag protein, indicative of impaired particle assembly. IPMK KO compromised virus production to a greater extent than IPPK KO suggesting that IP5 promotes HIV-1 particle assembly in IPPK KO cells. HIV-1 particles released from infected IPPK or IPMK KO cells were less infectious than those from WT cells. These viruses exhibited partially cleaved Gag proteins, decreased virion-associated p24, and higher frequencies of aberrant particles, indicative of a maturation defect. Our data demonstrate that IP6 enhances the quantity and quality of virions produced from T cells, thereby preventing defects in HIV-1 replication.     

Nanoscale orientation and lateral organization of chimeric metal-binding green fluorescent protein on lipid membrane determined by epifluorescence and atomic force microscopy

Epifluorescence microscopy as well as atomic force microscopy was successfully applied to explore the orientation and lateral organization of a group of chimeric green fluorescent proteins (GFPs) on lipid membrane. Incorporation of the chimeric GFP carrying Cd-binding region (His6CdBP4GFP) to the fluid phase of DPPC monolayer resulted in a strong fluorescence intensity at the air-water interface. Meanwhile, non-specific adsorption of the GFP having hexahistidine (His6GFP) led to the perturbation of the protein structure in which very low fluorescence was observed. Specific binding of both of the chimeric GFPs to immobilized zinc ions underneath the metal-chelating lipid membrane was revealed. This specific binding could be reversibly controlled by addition of metal ions or metal chelator. Binding of the chimeric GFPs to the metal-chelating lipid membrane was proven to be the end-on orientation while the side-on adsorption was contrarily noted in the absence of metal ions. Increase of lateral mobility owing to the fluidization effect on the chelating lipid membrane subsequently facilitated crystal formation. All these findings have opened up a potential approach for a specific orientation of immobilization of protein at the membrane interface. This could have accounted for a better opportunity of sensor development.     

Rapid Isolation of a Single-Chain Antibody against the Cyanobacterial Toxin Microcystin-LR by Phage Display and Its Use in the Immunoaffinity Concentration of Microcystins from Water

A naïve (unimmunized) human semisynthetic phage display library was employed to isolate recombinant antibody fragments against the cyanobacterial hepatotoxin microcystin-LR. Selected antibody scFv genes were cloned into a soluble expression vector and expressed in Escherichia coli for characterization against purified microcystin-LR by competition enzyme-linked immunosorbent assay (ELISA). The most sensitive single-chain antibody (scAb) isolated was capable of detecting microcystin-LR at levels below the World Health Organization limit in drinking water (1 μg liter⁻¹) and cross-reacted with three other purified microcystin variants (microcystin-RR, -LW, and -LF) and the related cyanotoxin nodularin. Extracts of the cyanobacterium Microcystis aeruginosa were assayed by ELISA, and quantifications of microcystins in toxic samples showed good correlation with analysis by high-performance liquid chromatography. Immobilized scAb was also used to prepare immunoaffinity columns, which were assessed for the ability to concentrate microcystin-LR from water for subsequent analysis by high-performance liquid chromatography. Anti-microcystin-LR scAb was immobilized on columns via a hexahistidine tag, ensuring maximum exposure of antigen binding sites, and the performance of the columns was evaluated by directly applying 150 ml of distilled water spiked with 4 μg of purified microcystin-LR. The procedure was simple, and a recovery rate of 94% was achieved following elution in 1 ml of 100% methanol. Large-scale, low-cost production of anti-microcystin-LR scAb in E. coli is an exciting prospect for the development of biosensors and on-line monitoring systems for microcystins and will also facilitate a range of immunoaffinity applications for the cleanup and concentration of these toxins from environmental samples.     

Scavenger receptor class B type I-mediated cholesteryl ester-selective uptake and efflux of unesterified cholesterol. Influence of high density lipoprotein size and structure

Scavenger receptor (SR)-BI catalyzes the selective uptake of cholesteryl ester (CE) from high density lipoprotein (HDL) by a two-step process that involves the following: 1) binding of HDL to the receptor and 2) diffusion of the CE molecules into the cell plasma membrane. We examined the effects of the size of discoidal HDL particles containing wild-type (WT) apoA-I on selective uptake of CE and efflux of cellular free (unesterified) cholesterol (FC) from COS-7 cells expressing SR-BI to determine the following: 1) the influence of apoA-I conformation on the lipid transfer process, and 2) the contribution of receptor binding-dependent processes to the overall efflux of cellular FC. Large (10 nm diameter) reconstituted HDL bound to SR-BI better (B(max) approximately 420 versus 220 ng of apoA-I/mg cell protein), delivered more CE, and promoted more FC efflux than small ( approximately 8 nm) particles. When normalized to the number of reconstituted HDL particles bound to the receptor, the efficiencies of either CE uptake or FC efflux with these particles were the same indicating that altering the conformation of WT apoA-I modulates binding to the receptor (step 1) but does not change the efficiency of the subsequent lipid transfer (step 2); this implies that binding induces an optimal alignment of the WT apoA-I.SR-BI complex so that the efficiency of lipid transfer is always the same. FC efflux to HDL is affected both by binding of HDL to SR-BI and by the ability of the receptor to perturb the packing of FC molecules in the cell plasma membrane.     

HEHEHE-tagged affibody molecule may be purified by IMAC, is conveniently labeled with [⁹⁹(m)Tc(CO)₃](+), and shows improved biodistribution with reduced hepatic radioactivity accumulation

Affibody molecules are a class of small (ca. 7 kDa) robust scaffold proteins suitable for radionuclide molecular imaging of therapeutic targets in vivo. A hexahistidine tag at the N-terminus streamlines development of new imaging probes by enabling facile purification using immobilized metal ion affinity chromatography (IMAC), as well as convenient [??(m)Tc(CO)?](+)-labeling. However, previous studies in mice have demonstrated that Affibody molecules labeled by this method yield higher liver accumulation of radioactivity, compared to the same tracer lacking the hexahistidine tag and labeled by an alternative method. Two variants of the HER2-binding Affibody molecule Z(HER)?(:)??? were made in an attempt to create a tagged tracer that could be purified by immobilized metal affinity chromatography, yet would not result in anomalous hepatic radioactivity accumulation following labeling with [??(m)Tc(CO)?](+). In one construct, the hexahistidine tag was moved to the C-terminus. In the other construct, every second histidine residue in the hexahistidine tag was replaced by the more hydrophilic glutamate, resulting in a HEHEHE-tag. Both variants, denoted Z(HER)?(:)???-H? and (HE)?-Z(HER)?(:)???, respectively, could be efficiently purified using IMAC and stably labeled with [??(m)Tc(CO)?](+) and were subsequently compared with the parental H?-Z(HER)?(:)??? having an N-terminal hexahistidine tag. All three variants were demonstrated to specifically bind to HER2-expressing cells in vitro. The hepatic accumulation of radioactivity in a murine model was 2-fold lower with [??(m)Tc(CO)?](+)-Z(HER2:342)-H? compared to [??(m)Tc(CO)?](+)-H?-Z(HER)?(:)???, and more than 10-fold lower with [??(m)Tc(CO)?](+)-(HE)?-Z(HER)?(:)???. These differences translated into appreciably superior tumor-to-liver ratio for [??(m)Tc(CO)?](+)-(HE)?-Z(HER)?(:)??? compared to the alternative conjugates. This information might be useful for development of other scaffold-based molecular imaging probes.     

Identification of candidate carrier proteins for surface display on Lactococcus lactis by theoretical and experimental analyses of the surface proteome

Lactococcus lactis is a lactic acid bacterium of proven safety for use in human oral applications. For this purpose, surface display of recombinant proteins is important, and new approaches for it are being sought. Analysis of the bacterial surface proteome is essential in identifying new candidate carrier proteins for surface display. We have made two different predictions of surface-associated proteins of L. lactis MG1363 by using Augur and LocateP software, which yielded 666 and 648 proteins, respectively. Surface proteins of L. lactis NZ9000, a derivative of MG1363, were identified by using a proteomics approach. The surface proteins were cleaved from intact bacteria, and the resulting peptides were identified by mass spectrometry. The latter approach yielded 80 proteins, 34 of which were not predicted by either software. Of the 80 proteins, 7 were selected for further study. These were cloned in frame with a C-terminal hexahistidine tag and overexpressed in L. lactis NZ9000 using nisin-controlled expression. Proteins of correct molecular weight carrying a hexahistidine tag were detected. Their surface localization was confirmed with flow cytometry. Basic membrane protein A (BmpA) was exposed at the highest level. To test BmpA as a candidate carrier protein, the hexahistidine tag was replaced by the B domain of staphylococcal protein A in the genetic construct. The B domain was displayed on the surface with BmpA as a carrier. The advantage of covalent BmpA binding was demonstrated. BmpA was thus shown to be a suitable candidate for a carrier protein in lactococcal surface display.     

Use of Algae for Removing Heavy Metal Ions From Wastewater: Progress and Prospects

ABSTRACT

Many algae have immense capability to sorb metals, and there is considerable potential for using them to treat wastewaters. Metal sorption involves binding on the cell surface and to intracellular ligands. The adsorbed metal is several times greater than intracellular metal. Carboxyl group is most important for metal binding. Concentration of metal and biomass in solution, pH, temperature, cations, anions and metabolic stage of the organism affect metal sorption. Algae can effectively remove metals from multi-metal solutions. Dead cells sorb more metal than live cells. Various pretreatments enhance metal sorption capacity of algae. CaCl₂ pretreatment is the most suitable and economic method for activation of algal biomass. Algal periphyton has great potential for removing metals from wastewaters. An immobilized or granulated biomass-filled column can be used for several sorption/desorption cycles with unaltered or slightly decreased metal removal. Langmuir and Freundlich models, commonly used for fitting sorption data, cannot precisely describe metal sorption since they ignore the effect of pH, biomass concentration, etc. For commercial application of algal technology for metal removal from wastewaters, emphasis should be given to: (i) selection of strains with high metal sorption capacity, (ii) adequate understanding of sorption mechanisms, (iii) development of low-cost methods for cell immobilization, (iv) development of better models for predicting metal sorption, (v) genetic manipulation of algae for increased number of surface groups or over expression of metal binding proteins, and (vi) economic feasibility.     

Novel Surface Display System for Proteins on Non-Genetically Modified Gram-Positive Bacteria

A novel display system is described that allows highly efficient immobilization of heterologous proteins on bacterial surfaces in applications for which the use of genetically modified bacteria is less desirable. This system is based on nonliving and non-genetically modified gram-positive bacterial cells, designated gram-positive enhancer matrix (GEM) particles, which are used as substrates to bind externally added heterologous proteins by means of a high-affinity binding domain. This binding domain, the protein anchor (PA), was derived from the Lactococcus lactis peptidoglycan hydrolase AcmA. GEM particles were typically prepared from the innocuous bacterium L. lactis, and various parameters for the optimal preparation of GEM particles and binding of PA fusion proteins were determined. The versatility and flexibility of the display and delivery technology were demonstrated by investigating enzyme immobilization and nasal vaccine applications.