Affinity Chromatography Method Research Articles

Biodegradation of poly(3‐hydroxybutyrate‐co‐4‐hydroxybutyrate) by a novel P3/4HB depolymerase purified from Agrobacterium sp. DSGZ

ABSTRACTA poly(3‐hydroxybutyrate‐co‐4‐hydroxybutyrate) (P3/4HB)‐degrading strain, Agrobacterium sp. DSGZ, was isolated from sewage by poly(3‐hydroxybutyrate) (PHB) mineral agar plates. A novel P3/4HB depolymerase with a molecular weight of 34 kDa was purified through a novel single‐step affinity chromatography method from the culture supernatant of the strain by using P3/4HB powder as a substrate. The purified depolymerase showed optimum activity at pH 7.0 and 50°C, and was stable at the pH range of 6.0 to 9.0 and temperature below 50°C. Enzyme activity was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), ethylenediaminetetraacetic acid (EDTA), hydrophobic reagents, and some metal ions. The depolymerase degraded poly(3‐hydroxybutyrate) (PHB), poly(hydroxybutyrate‐co‐hydroxyvalerate) (PHBV), P3/4HB, and polycaprolactone (PCL), instead of polylactic acid (PLA) or poly(butylene succinate) (PBS). Meanwhile, the depolymerase showed high hydrolytic activity against short‐chain length esters, such as butyrate acid ester and caprylic acid ester. The main degradation products of the depolymerase were identified as hydroxybutyrate monomers and dimers, and the monomers were identified as 3‐hydroxybutyrate (3HB) monomers and 4‐hydroxybutyrate (4HB) monomers. The preparation procedure, crystallinity, and 4HB composition of the P3/4HB copolymer showed evident effect on degradation behavior, and change in crystallinity was the main factor affecting degradation. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016, 133, 42805.

Complementary IMAC enrichment methods for HLA-associated phosphopeptide identification by mass spectrometry.

Phosphorylation events within cancer cells often become dysregulated, leading to oncogenic signaling and abnormal cell growth. Phosphopeptides derived from aberrantly phosphorylated proteins that are presented on tumors and not on normal tissues by human leukocyte antigen (HLA) class I molecules are promising candidates for future cancer immunotherapies, because they are tumor specific and have been shown to elicit cytotoxic T cell responses. Robust phosphopeptide enrichments that are suitable for low input amounts must be developed to characterize HLA-associated phosphopeptides from clinical samples that are limited by material availability. We present two complementary mass spectrometry-compatible, iron(III)-immobilized metal affinity chromatography (IMAC) methods that use either nitrilotriacetic acid (NTA) or iminodiacetic acid (IDA) in-house-fabricated columns. We developed these protocols to enrich for subfemtomole-level phosphopeptides from cell line and human tissue samples containing picograms of starting material, which is an order of magnitude less material than what is commonly used. In addition, we added a peptide esterification step to increase phosphopeptide specificity from these low-input samples. To date, hundreds of phosphopeptides displayed on melanoma, ovarian cancer, leukemia and colorectal cancer have been identified using these highly selective phosphopeptide enrichment protocols in combination with a program called 'CAD Neutral Loss Finder' that identifies all spectra containing the characteristic neutral loss of phosphoric acid from phosphorylated serine and threonine residues. This methodology enables the identification of HLA-associated phosphopeptides presented by human tissue samples containing as little as nanograms of peptide material in 2 d.

Clinically relevant lot-to-lot reagent difference in a commercial immunoturbidimetric assay for glycated hemoglobin A1c

ObjectivesHemoglobin A1c (HbA1c) is employed for diagnosis and therapy monitoring of diabetes mellitus. The effect of a change of reagent lot on the measured values of a commercial immunoturbidimetric HbA1c assay (A1C3) was investigated. Design and methodsComparison measurements of A1C3 and an automated affinity chromatography method (VIIT) were performed in 15 samples for the initial and in 20 samples for the subsequent A1C3 lot. The results of 27 and 19 measurements of a normal and of 28 and 20 of a pathological control (before and after the switch of the A1C3 reagent lot, respectively) were evaluated. Finally, the results of 6463 patient samples that had been measured with the initial and 434 that had been measured with the subsequent A1C3 lot were investigated. ResultsVIIT yielded significantly higher results than the initial A1C3 lot (bias: 0.41% HbA1c, 4.5mmol/mol) but agreed well with the subsequent lot (bias: −0.01% HbA1c, −0.1mmol/mol). Changing to the subsequent reagent lot resulted in significant increases of the mean of the normal control of 0.316% HbA1c (3.5mmol/mol) and of the pathological control of 0.749% HbA1c (8.2mmol/mol). The median of patient samples measured with the subsequent lot was significantly higher by 0.40% HbA1c (4.4mmol/mol). ConclusionsThe subsequent A1C3 reagent lot yields significantly higher measurement results than the initial by approximately 0.5% HbA1c (5.5mmol/mol). This difference is considered as clinically relevant. A combined effort of manufacturers and notified bodies is necessary to minimize lot-to-lot variation.

Development of an aptamer-affinity chromatography for efficient single step purification of Concanavalin A from Canavalia ensiformis.

Herein, an aptamer-based affinity chromatography method for rapid and single step purification of Concanavalin A is developed and validated. We have used a 41ntssDNA aptamer of Con A (Con A aptabody) as an affinity reagent in the developed aptamer-affinity chromatography. Stationary phase of the method consists of surface functionalized agarose beads carrying covalently immobilized Con A-aptabody. Affinity purification of Con A from jack bean (Canavalia ensiformis) seed using developed aptamer-affinity columns has resulted in ≥66% recovery with 90% purity and 336-fold purification of Con A. The developed aptamer-affinity chromatography has shown efficient scalability and consistent purification when analysed over 13mm, 20mm and 25mm diameter columns having a bed height of 60mm each. Also, the developed aptamer-agarose columns were found to be reusable with recovery decrease of 12.9% in seven sequential cycles of purification. Therefore, the developed aptamer-affinity chromatography provides a novel, efficient and single-step methodology for isolation and purification of Con A.

EXPRESSION OF b-XYLOSIDASE ENCODING GENE IN PHIS/ Bacillus megaterium MS SYSTEM

b-Xylosidase encoding gene from G. thermoleovorans IT-08 had been expressed in the pHIS1525/ B. megaterium MS941 system. The b-xylosidase gene (xyl) was inserted into plasmid pHIS1525 and propagated in E. coli DH10b. The recombinant plasmid was transformed into B. megaterium MS941 by protoplast transformation. Transformants were selected by growing the recombinant cells on solid LB medium containing tetracycline (10 µg/ ml). The expression of the b-xylosidase gene was assayed by overlaid the recombinant B. megaterium MS941 cell with agar medium containing 0.2% ethylumbelliferyl-b-D-xyloside (MUX). This research showed that the b-xylosidase gene was succesfully sub-cloned in pHIS1525 system and expressed by the recombinant B. megaterium MS941. Theaddition of 0.5% xylose into the culture medium could increase the activity of recombinantactivity of recombinant of recombinantb-xylosidase by 2.74 fold. The recombinant B. megaterium MS941 secreted 75.56% of the expressed b-xylosidase into culture medium. The crude extract b-xylosidase showed the optimum activity at 50° C and pH 6. The recombinant b-xylosidase was purified from culture supernatant by affinity chromatographic method using agarose containing Ni-NTA (Nickel-Nitrilotriacetic acid). The pure b-xylosidase showed a specific activity of 10.06 Unit/mg protein and relative molecular weight ± 58 kDa.

Efficient soluble expression of active recombinant human cyclin A2 mediated by E. coli molecular chaperones

Bacterial expression of human proteins continues to present a critical challenge in protein crystallography and drug design. While human cyclin A constructs have been extensively characterized in complex with cyclin dependent kinase 2 (CDK2), efforts to express the monomeric human cyclin A2 in Escherichia coli in a stable form, without the kinase subunit, have been laden with technical difficulties, including solubility, yield and purity. Here, optimized conditions are described with the aim of generating for first time, sufficient quantities of human recombinant cyclin A2 in a soluble and active form for crystallization and ligand characterization purposes. The studies involve implementation of a His-tagged heterologous expression system under conditions of auto-induction and mediated by molecular chaperone-expressing plasmids. A high yield of human cyclin A2 was obtained in natively folded and soluble form, through co-expression with groups of molecular chaperones from E. coli in various combinations. A one-step affinity chromatography method was utilized to purify the fusion protein products to homogeneity, and the biological activity confirmed through ligand-binding affinity to inhibitory peptides, representing alternatives for the key determinants of the CDK2 substrate recruitment site on the cyclin regulatory subunit. As a whole, obtaining the active cyclin A without the CDK partner (referred to as monomeric in this work) in a straightforward and facile manner will obviate protein – production issues with the CDK2/cyclin A complex and enable drug discovery efforts for non-ATP competitive CDK inhibition through the cyclin groove.

Method for trapping affinity chromatography of transcription factors using aldehyde–hydrazide coupling to agarose

The use of a method of coupling DNA was investigated for trapping and purifying transcription factors. Using the GFP–C/EBP (CAAT/enhancer binding protein) fusion protein as a model, trapping gives higher purity and comparable yield to conventional affinity chromatography. The chemistry used is mild and was shown to have no detrimental effect on GFP fluorescence or GFP–C/EBP DNA binding. The method involves introducing a ribose nucleotide to the 3′ end of a DNA sequence. Reaction with mM NaIO4 (sodium metaperiodate) produces a dialdehyde of ribose that couples to hydrazide–agarose. The DNA is combined at nM concentration with a nuclear extract or other protein mixture, and DNA–protein complexes form. The complex is then coupled to hydrazide–agarose for trapping the DNA–protein complex and the protein eluted by increasing NaCl concentration. Using a different oligonucleotide with the proximal E-box sequence from the human telomerase promoter, USF-2 transcription factor was purified by trapping, again with higher purity than results from conventional affinity chromatography and similar yield. Other transcription factors binding E-boxes, including E2A, c-Myc, and Myo-D, were also purified, but myogenin and NFκB were not. Therefore, this approach proved to be valuable for both affinity chromatography and the trapping approach.

The small G protein Arl5 contributes to endosome-to-Golgi traffic by aiding the recruitment of the GARP complex to the Golgi.

The small G proteins of the Arf family play critical roles in membrane trafficking and cytoskeleton organization. However, the function of some members of the family remains poorly understood including Arl5 which is widely conserved in eukaryotes. Humans have two closely related Arl5 paralogues (Arl5a and Arl5b), and both Arl5a and Arl5b localize to the trans-Golgi with Arl5b being involved in retrograde traffic from endosomes to the Golgi apparatus. To investigate the function of Arl5, we have used Drosophila melanogaster as a model system. We find that the single Arl5 orthologue in Drosophila also localizes to the trans-Golgi, but flies lacking the Arl5 gene are viable and fertile. By using both liposome and column based affinity chromatography methods we find that Arl5 interacts with the Golgi-associated retrograde protein (GARP) complex that acts in the tethering of vesicles moving from endosomes to the trans-Golgi network (TGN). In Drosophila tissues the GARP complex is partially displaced from the Golgi when Arl5 is absent, and the late endosomal compartment is enlarged. In addition, in HeLa cells GARP also becomes cytosolic upon depletion of Arl5b. These phenotypes are consistent with a role in endosome-to-Golgi traffic, but are less severe than loss of GARP itself. Thus it appears that Arl5 is one of the factors that directs the recruitment of the GARP complex to the trans-Golgi, and this function is conserved in both flies and humans.

Arabidopsis glutaredoxin S17 and its partner, the nuclear factor Y subunit C11/negative cofactor 2α, contribute to maintenance of the shoot apical meristem under long-day photoperiod.

Glutaredoxins (GRXs) catalyze the reduction of protein disulfide bonds using glutathione as a reductant. Certain GRXs are able to transfer iron-sulfur clusters to other proteins. To investigate the function of Arabidopsis (Arabidopsis thaliana) GRXS17, we applied a strategy combining biochemical, genetic, and physiological approaches. GRXS17 was localized in the nucleus and cytosol, and its expression was elevated in the shoot meristems and reproductive tissues. Recombinant GRXS17 bound Fe2S2 clusters, a property likely contributing to its ability to complement the defects of a Baker's yeast (Saccharomyces cerevisiae) strain lacking the mitochondrial GRX5. However, a grxs17 knockout Arabidopsis mutant exhibited only a minor decrease in the activities of iron-sulfur enzymes, suggesting that its primary function is as a disulfide oxidoreductase. The grxS17 plants were sensitive to high temperatures and long-day photoperiods, resulting in elongated leaves, compromised shoot apical meristem, and delayed bolting. Both environmental conditions applied simultaneously led to a growth arrest. Using affinity chromatography and split-Yellow Fluorescent Protein methods, a nuclear transcriptional regulator, the Nuclear Factor Y Subunit C11/Negative Cofactor 2α (NF-YC11/NC2α), was identified as a GRXS17 interacting partner. A mutant deficient in NF-YC11/NC2α exhibited similar phenotypes to grxs17 in response to photoperiod. Therefore, we propose that GRXS17 interacts with NF-YC11/NC2α to relay a redox signal generated by the photoperiod to maintain meristem function.

Purification and characterization of polyphenol oxidase enzyme from Iğdır apricot (Prunus armeniaca L.)

In this study, polyphenol oxidase (E.C. 1.14.18.1; PPO) enzyme obtained from Igdir Apricot was purified with method of affinity chromatography. The apricot cultivar “Salak ” was provided from Igdir region. To purify polyphenol oxidase enzyme obtained

Properties and some in vitro studies of 6-phosphogluconate dehydrogenase purified from the liver of Chalcalburnus tarichi, the only fish living in Lake Van's highly alkaline water (pH 9.8)

In this study, some enzymology parameters and some antibiotics affecting the enzyme activity of 6-phosphogluconate dehydrogenase (6PGD) extracted from t the livers of fish from Lake Van (\textit{Chalcalburnus tarichi}) were investigated because it is an important enzyme-producing NADPH, a reductive power, protecting the cell against the oxidative agents by producing reduced glutathione. The crude enzyme solution was obtained by using the affinity chromatography method. The native molecular weight of the enzyme is $\sim $90,000 $\pm $ 3000 Da and it is composed of two subunits with identical molecular weights of $\sim $46,000 Da as exhibited on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The pH and temperature for optimal conditions of 6PGD were about 8.5 and 40 $^{\circ}$C, respectively, and the enzyme showed optimal activity in 40 mM ammonium sulfate solution. In addition, in vitro effects of clindamycin phosphate, streptomycin sulfate, and lincomycin antibiotics on the enzyme activity were investigated.

Dengue virus type-3 envelope protein domain III; expression and immunogenicity.

Production of a recombinant and immunogenic antigen using dengue virus type-3 envelope protein is a key point in dengue vaccine development and diagnostic researches. The goals of this study were providing a recombinant protein from dengue virus type-3 envelope protein and evaluation of its immunogenicity in mice. Multiple amino acid sequences of different isolates of dengue virus type-3, corresponding to the envelope protein domain III, were achieved from GenBank. Clustal V alignment tool was used to provide a consensus amino acid sequence. Nucleotide sequence of the coding gene was optimized using "Optimizer". The origami (DE3) strain of Escherichia coli was used as the host in order to express the protein. A commercial affinity chromatography method was used to purify the recombinant protein. Immunogenicity of the recombinant protein was evaluated in mice using ELISA, MTT and cytokine assays. A consensus amino acid sequence corresponding to the most important region of dengue virus type-3 envelope protein (domain III) was provided. A high concentration (≥ 20 mg/L culture medium) of soluble recombinant antigen (EDIII3) was achieved. Immunized mice developed specific antibody responses against EDIII3 protein. The splenocytes from EDIII3-immunized mice showed a high proliferation rate in comparison with the negative control. In addition, the concentrations of two measured cytokines (IFN-γ and IL-4) were increased markedly in immunized mice. The results showed that the expressed recombinant EDIII3 protein is an immunogenic antigen and can be applied to induce specific immune responses against dengue virus type-3.

Pharmaceutical-grade pre-miR-29 purification using an agmatine monolithic support

MicroRNA-based therapeutic applications have fostered a growing interest in the development of microRNAs purification processes in order to obtain the final product with high purity degree, good quality and biologically active. The pre-miR-29 deficiency or overexpression has been associated to a number of clinically important diseases, and its therapeutic application can be considered. Monolithic columns emerged as a new class of chromatographic supports used in the plasmid DNA purification platforms, being an interesting alternative to the conventional particle-based columns. Thus, the current work describes, for the first time, a new affinity chromatography method that combines the high selectivity of agmatine ligands with the versatility of monoliths to specifically and efficiently purify pre-miR-29 from other small RNA species and Rhodovulum sulfidophilum impurities. The effect of different flow rates on pre-miR-29 separation was also evaluated. Moreover, breakthrough experiments were designed to study the effect of different RNA concentrations on the modified monolithic support binding capacity, being verified that the dynamic binding capacity for RNA molecules is dependent of the feed concentration. In order to achieve higher efficiency and selectivity, three different binding and elution strategies based on increased sodium chloride (1.75–3M) or arginine (100mM) and decreased ammonium sulfate (2.4–0M) stepwise gradients are described to purify pre-miR-29. As a matter of fact, by employing elution strategies using sodium chloride or arginine, an improvement in the final pre-miR-29 yields (97.33 and 94.88%, respectively) as well as purity (75.21 and 90.11%, respectively) were obtained. Moreover, the quality control analysis revealed that the level of impurities (proteins, endotoxins, sRNA) in the final pre-miR-29 sample was negligible. In fact, this new monolithic support arises as a powerful instrument on the microRNA purification to be used in further clinical applications, providing a more rapid and economical purification platform.

Switchable aptamers for biosensing and bioseparation of viruses (SwAps-V).

There is a widespread interest in the development of aptamer-based affinity chromatographic methods for purification of biomolecules. Regardless of the many advantages exhibited by aptamers when compared to other recognition elements, the lack of an efficient regeneration technique that can be generalized to all targets has encumbered further integration of aptamers into affinity-based purification methods. Here we offer switchable aptamers (SwAps) that have been developed to solve this problem and move aptamer-based chromatography forward. SwAps are controlled-affinity aptamers, which have been employed here to purify vesicular stomatitis virus (VSV) as a model case, however this technique can be extended to all biologically significant molecules. VSV is one oncolytic virus out of an arsenal of potential candidates shown to provide selective destruction of cancer cells both in vitro and in vivo. These SwAps were developed in the presence of Ca(2+) and Mg(2+) ions where they cannot bind to their target VSV in absence of these cations. Upon addition of EDTA and EGTA, the divalent cations were sequestered from the stabilized aptameric structure causing a conformational change and subsequently release of the virus. Both flow cytometry and electrochemical impedance spectroscopy were employed to estimate the binding affinities between the selected SwAps and VSV and to determine the coefficient of switching (CoS) upon elution. Among fifteen sequenced SwAps, four have exhibited high affinity to VSV and ability to switch upon elution and thus were further integrated into streptavidin-coated magnetic beads for purification of VSV.

Quality improvements of cell membrane chromatographic column

Cell Membrane Chromatography (CMC) is a biological affinity chromatographic method using a silica stationary phase covered with specific cell membrane. However, its short life span and poor quality control was highlighted in a lot of research articles. In this study, special attention has been paid to the disruption, cell load and packing procedure in order to improve the quality of the CMC columns. Hereto, two newly established CMC models, HSC-T6/CMC and SMMC-7721/CMC have been developed and used in this research project. The optimization of the abovementioned parameters resulted in a better reproducibility of the retention time of the compound GFT (RSD<10%) and improved significantly the quality of the CMC columns. 3.5×107cells were the optimal cell load for the preparation of the CMC columns, the disruption condition was optimized to 5 cycles (400W and 20s interval per cycle) by an ultrasonic processor reducing the total time of cell disruption to 1.5min and the packing flow rate was optimized by applying a linear gradient program. Additionally, 4% paraformaldehyde (PFA) was employed to improve the column quality and prolong the column life span. The results showed that the retention time was longer with PFA treated columns than the ones obtained with the control groups

AB0158 Deoxyribonuclease Activity of Polyclonal Iggs – A Putative Serological Marker of Autoimmune Inflammation in Patients with Spondyloarthritides

BackgroundAntibodies executing catalytic activity are referred to as antibody enzymes or short “abzymes” and may have diagnostic relevance. Abzymes with deoxyribonuclease (DNase) activity have been demonstrated in patients with autoimmune...

English

Cytotoxicity of tuber lectins from two potato cultivars was assessed and their anti-tumor potential against experimentally induced Ehrlich ascites carcinoma in Swiss albino mice was evaluated. Twenty (20) kDa chitin-binding lectins from Solanum tuberosum tubers, STL-S and STL-D were purified through ion-exchange and affinity chromatographic methods, hemagglutinating activity and blood group specificity of the lectins were checked whereas the cytotoxicity was determined using brine shrimp (Artemia salina L.) nauplii lethality assay. The lectins showed no specificity to animal and human erythrocytes. LC50 values for STL-S and STL-D were found to be 75 and 90 &mu;g/ml, respectively with a dose-dependent intermediary toxic effect. After inducing ascites by intraperitoneal propagation, the Swiss albino mice were treated by administering the lectins at a dose of 1.38 mg/kg/day for five consecutive days. STL-S and STL-D showed 79.84 and 83.04% of growth inhibition of EAC cells, respectively. Additionally, hemoglobin and RBC levels became considerably increased with a drop off in the WBC levels in the treated mice group indicating moderate anticancer activities exhibited by the potato lectins. &nbsp; Key words: Chitin-binding lectins, antitumor activity, LC50, cell growth inhibition.

Cloning, expression and purification of recombinant Slx4 from Saccharomyces cerevisiae (539.3)

Cells are constantly exposed to exogenous and endogenous sources of DNA damage. In the presence of genotoxic stress, the DNA damage checkpoint signaling (DDC) pathway contributes to genomic maintenance by promoting a transient cell cycle arrest and recruiting numerous repair proteins to sites of lesions, thus maintaining genomic integrity. Slx4 is an evolutionary conserved scaffold protein that is important for the cellular response to genotoxic stress. Slx4 interacts with several DNA repair factors and also with components of the DDC. Recently, our group showed that Slx4 in Saccharomyces cerevisiae is capable to interact with a DNA replication initiation protein called Dpb11, therefore dampening the checkpoint by counteracting the Rad9 adaptor protein. This competition based mechanism is important to avoid hyperactivation of the DDC which can be deleterious to the cells. In order to better understand the role of Slx4 during DDC, we decided to purify the protein and to elucidate its structure by X‐ray diffraction. Our hypothesis was that by employing the method of metal affinity chromatography using cobalt a resin we would be able to purify our Slx4 protein. Here, we cloned, expressed and purified recombinant Slx4 by heterologous expression in Escherichia coli. We cloned a truncated form of the Slx4 gene (361aa to the end) into an expression vector pET28a. We transformed the product into an E. coli expression strain Bl21 (DE3) that contains the T7 RNA polymerase to express the Slx4 gene through the T7 promoter from the pET28a vector. To induce the expression of the Slx4 gene, we added IPTG to further purify the protein. In conclusion we were able to express Slx4 (361aa‐end) in the soluble fraction of the cell and purify in its native form through metal affinity chromatography using cobalt resin.Grant Funding Source: R25GM096955

PDZ affinity chromatography: A general method for affinity purification of proteins based on PDZ domains and their ligands

PDZ (PSD-95, DiscsLarge, ZO1) domains function in nature as protein binding domains within scaffold and membrane-associated proteins. They comprise ∼90 residues and make specific, high affinity interactions with complementary C-terminal peptide sequences, with other PDZ domains, and with phospholipids. We hypothesized that the specific, strong interactions of PDZ domains with their ligands would make them well suited for use in affinity chromatography. Here we describe a novel affinity chromatography method applicable for the purification of proteins that contain PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We created a series of affinity resins comprised of PDZ domains from the scaffold protein PSD-95, or from neuronal nitric oxide synthase (nNOS), coupled to solid supports. We used them to purify heterologously expressed neuronal proteins or protein domains containing endogenous PDZ domain ligands, eluting the proteins with free PDZ domain peptide ligands. We show that Proteins of Interest (POIs) lacking endogenous PDZ domain ligands can be engineered as fusion products containing C-terminal PDZ domain ligand peptides or internal, N- or C-terminal PDZ domains and then can be purified by the same method. Using this method, we recovered recombinant GFP fused to a PDZ domain ligand in active form as verified by fluorescence yield. Similarly, chloramphenicol acetyltransferase (CAT) and β-Galactosidase (LacZ) fused to a C-terminal PDZ domain ligand or an N-terminal PDZ domain were purified in active form as assessed by enzymatic assay. In general, PDZ domains and ligands derived from PSD-95 were superior to those from nNOS for this method. PDZ Domain Affinity Chromatography promises to be a versatile and effective method for purification of a wide variety of natural and recombinant proteins.

Proteomic analysis of copper-binding proteins in excess copper-stressed rice roots by immobilized metal affinity chromatography and two-dimensional electrophoresis

Copper (Cu) is an essential micronutrient required for plant growth and development. However, excess Cu can inactivate and disturb protein structure as a result of unavoidable binding to proteins. To understand better the mechanisms involved in Cu toxicity and tolerance in plants, we developed a new immobilized metal affinity chromatography (IMAC) method for the separation and isolation of Cu-binding proteins extracted from roots of rice seedling exposed to excess Cu. In our method, IDA-Sepharose or EDDS-Sepharose column (referred as pre-chromatography) and Cu-IDA-Sepharose column (referred as Cu-IMAC) were connected in tandem. Namely, protein samples were pre-chromatographed with IDA-Sepharose column to removal metal ions, then protein solution was flowed into Cu-IMAC column for enriching Cu-binding proteins in vitro. Compared with the control (Cu-IMAC without any pre-chromatography), IDA-Sepharose pre-chromatography method markedly increased yield of the Cu-IMAC-binding proteins, and number of protein spots and the abundance of 40 protein spots on two-dimensional electrophoresis (2-DE) gels. Thirteen protein spots randomly selected from 2-DE gel and 11 proteins were identified using MALDI-TOF-TOF MS. These putative Cu-binding proteins included those involved in antioxidant defense, carbohydrate metabolism, nucleic acid metabolism, protein folding and stabilization, protein transport and cell wall synthesis. Ten proteins contained one or more of nine putative metal-binding motifs reported by Smith et al. (J Proteome Res 3:834-840, 2004) and seven proteins contained one or two of top six motifs reported by Kung et al. (Proteomics 6:2746-2758, 2006). Results demonstrated that more proteins specifically bound with Cu-IMAC could be enriched through removal of metal ions from samples by IDA-Sepharose pre-chromatography. Further studies are needed on metal-binding characteristics of these proteins in vivo and the relationship between Cu ions and protein biological activities to fully understand the mechanisms of Cu tolerance and toxicity in plants.