Affinity Resin Research Articles

Novel Feruloyl Esterase from Lactobacillus fermentum NRRL B-1932 and Analysis of the Recombinant Enzyme Produced in Escherichia coli.

A total of 33 Lactobacillus strains were screened for feruloyl esterase (FE) activity using agar plates containing ethyl ferulate as the sole carbon source, and Lactobacillus fermentum NRRL B-1932 demonstrated the strongest FE activity among a dozen species showing a clearing zone on the opaque plate containing ethyl ferulate. FE activities were monitored using high-performance liquid chromatography with an acetonitrile-trifluoroacetic acid gradient. To produce sufficient purified FE from L. fermentum strain NRRL B-1932 (LfFE), the cDNA encoding LfFE (Lffae) was amplified and cloned by using available closely related genome sequences and overexpressed in Escherichia coli A 29.6-kDa LfFE protein was detected from the protein extract of E. coli BL21(pLysS) carrying pET28bLffae upon IPTG (isopropyl-β-d-thiogalactopyranoside) induction. The recombinant LfFE containing a polyhistidine tag was purified by nickel-nitrilotriacetic acid affinity resin. The purified LfFE showed strong activities against several artificial substrates, including p-nitrophenyl acetate and 4-methylumbelliferyl p-trimethylammoniocinnamate chloride. The optimum pH and temperature of the recombinant LfFE were around 6.5 and 37°C, respectively, as determined using either crude or purified recombinant LfFE. This study will be essential for the production of the LfFE in E. coli on a larger scale that could not be readily achieved by L. fermentum fermentation. The production of feruloyl esterase (FE) from Lactobacillus fermentum NRRL B-1932 reported in this study will have immense potential commercial applications not only in biofuel production but also in pharmaceutical, polymer, oleo chemical, cosmetic additive, and detergent industries, as well as human health-related applications, including food flavoring, functional foods, probiotic agents, preventive medicine, and animal feed. Given the essential role FE plays in the production of hydroxycinnamic acids and ferulic acid, plus the generally regarded as safe status of lactobacilli, which therefore have less regulatory concerns, LfFE from the probiotic L. fermentum reported in this work can be directly used for increased production of high-value hydroxycinnamates and ferulic acid from natural or synthetic carbon sources.

460. Purification of AAV8 Using a Prototype Affinity Resin Compatible with cGMP Manufacturing

Affinity interaction based resins have long been the foundation for various purification platforms of biological molecules. For the purification of rAAV, the affinity resin AVB Sepharose HP (AVB) has been used to successfully purify a number of different serotypes. However, to date the use of AVB for the purification of the AAV8 serotype has been relatively unsuccessful due in part to modest binding capacity. In this work we investigate the use of a new, prototype affinity resin for the purification of AAV8 from crude feed streams. Performance of the AVB and prototype resins were directly compared for the purification of selected AAV8 feed streams. Differences in capacities, loading steps, wash step tolerances and elution steps will be discussed. In addition, the resulting performance of purifying AAV8 using the prototype resin is directly compared against the best case performance of the AVB resin purifying a different serotype more commonly used on AVB. Finally, the scale-up use of the prototype resin from bench to manufacturing will be discussed. These results demonstrate that the prototype affinity resin is a viable option for the scaleable, chromatographic purification of AAV8 from crude harvest streams.

294. Characterizing AAV CaptureSelect Affinity Ligand Interactions

Adeno-associated virus (AAV) is one of the most widely studied viral vector systems for therapeutic gene delivery. This application has already experienced success in several human clinical trials, including the treatment of hemophilia B with an rAAV8 vector expressing therapeutic levels of Factor IX protein and for the treatment of Pompe disease, utilizing AAV1 vectors. Recently, an rAAV1 vector packaging a gene for the treatment of lipoprotein lipase deficiency was approved as the first gene therapy drug, validating the utility of this system. However, the effective production and purification of enough viral vector for use in research, preclinical studies, and human treatment is essential for success. Towards this goal, camelid single domain antibody fragments directed against AAV were used to make affinity resin products suitable for fast, one-step purification of AAV vectors of several serotypes. This affinity based purification method can replace current standards of density purification, which are time consuming and require large volumes of reagents, such as sucrose or iodixanol. At present, the collection of affinity resins suitable for AAV purification, i.e. AVB-Sepharose™, POROS™ CaptureSelect™ AAV8 and -AAV9, show varied affinity for different AAV serotypes, resulting in varied efficacies of virus purification. To better understand these differences and improve broad range utility, it is important to understand how these affinity ligands bind to the surface of the AAV capsids. We have thus mapped their binding footprints on several AAV serotypes, including AAV1, AAV5, and AAV9, using cryo-electron microscopy and image reconstruction combined with pseudo-atomic modeling. The sites for different affinity ligands are clustered on regions of the capsid that are common among most serotypes, but display minor variations which likely account for the varied affinities. This information will assist in the synergistic development of affinity ligands with broader serotype coverage and AAV vectors with improved purification outcomes.

Engineered Aminoacyl-tRNA Synthetase for Cell-Selective Analysis of Mammalian Protein Synthesis.

Methods for cell-selective analysis of proteome dynamics will facilitate studies of biological processes in multicellular organisms. Here we describe a mutant murine methionyl-tRNA synthetase (designated L274GMmMetRS) that charges the noncanonical amino acid azidonorleucine (Anl) to elongator tRNAMet in hamster (CHO), monkey (COS7), and human (HeLa) cell lines. Proteins made in cells that express the synthetase can be labeled with Anl, tagged with dyes or affinity reagents, and enriched on affinity resin to facilitate identification by mass spectrometry. The method does not require expression of orthogonal tRNAs or depletion of canonical amino acids. Successful labeling of proteins with Anl in several mammalian cell lines demonstrates the utility of L274GMmMetRS as a tool for cell-selective analysis of mammalian protein synthesis.

Preparation and Affinity-Purification of Supervillin Isoform 4 (SV4) Specific Polyclonal Antibodies.

Human Supervillin isoform 4 (SV4), a bigger splicing isoform of Supervillin, contains extra coding exons 3, 4 and 5 (E345), compared to Supervillin isoform 1. Although previous studies have shown that SV4 associated with membrane and cytoskeleton, regulated cell migration and cell survival, its functions are still largely unknown. To broaden our understanding, SV4 specific antibody is important for further study in signaling pathway. The His-SV4 (E345) and GST-SV4 (E345) fusion proteins, which contained SV4 specific domain E345, were purified from bacteria. The His-SV4 (E345) proteins were injected in rabbits as immunogen to produce anti-SV4 serum, and SV4 antibodies were purified by GST-SV4 (E345) proteins cross-linked to affinity resins. SV4 antibodies exclusively recognized SV4 protein both in vitro and in vivo through multi-step testing by ELISA, western blot, immunoprecipitation, and immunofluorescence. Taken together, our data demonstrate a novel SV4-specific polyclonal antibody which will provide a useful tool for further characterization of SV4 function.

A Novel Recombinant DNA System for High Efficiency Affinity Purification of Proteins in Saccharomyces cerevisiae.

Isolation of endogenous proteins from Saccharomyces cerevisiae has been facilitated by inserting encoding polypeptide affinity tags at the C-termini of chromosomal open reading frames (ORFs) using homologous recombination of DNA fragments. Tagged protein isolation is limited by a number of factors, including high cost of affinity resins for bulk isolation and low concentration of ligands on the resin surface, leading to low isolation efficiencies and trapping of contaminants. To address this, we have created a recombinant “CelTag” DNA construct from which PCR fragments can be created to easily tag C-termini of S. cerevisiae ORFs using selection for a nat1 marker. The tag has a C-terminal cellulose binding module to be used in the first affinity step. Microgranular cellulose is very inexpensive and has an effectively continuous ligand on its surface, allowing rapid, highly efficient purification with minimal background. Cellulose-bound proteins are released by specific cleavage of an included site for TEV protease, giving nearly pure product. The tag can be lifted from the recombinant DNA construct either with or without a 13x myc epitope tag between the target ORF and the TEV protease site. Binding of CelTag protein fusions to cellulose is stable to high salt, nonionic detergents, and 1 M urea, allowing stringent washing conditions to remove loosely associated components, as needed, before specific elution. It is anticipated that this reagent could allow isolation of protein complexes from large quantities of yeast extract, including soluble, membrane-bound, or nucleic acid-associated assemblies.

Cleavage of fusion proteins on the affinity resins using the TEV protease variant

It is documented that the tobacco etch virus protease (TEVp) variant TEVp3M is less efficient in cleaving the fusion protein bound to Ni-NTA resin at relatively low temperature. Here, we determined that, using the GFP fusion substrate bound to Ni-NTA or Strep-tactin agarose, activity of the TEVp5M variant was higher than that of the other TEVp construct, and about 15% higher than that of the TEVp3M. The GST fusion proteins immobilized on Strep-tactin agarose or Glutathione Sepharose were efficiently cleaved by purified TEVp5M at specified conditions using GFP reporter for visual track and detection. After on-column cleavage of three fusion constructs using the cognate TEVp5M constructs, two target proteins with relatively high purity were separated from Ni-NTA or Amylose agarose. With elution of the buffer containing 1 M NaCl, maize sulfiredoxin was released from Ni-NTA resin via on-column cleavage. Our results confirmed that TEVp5M efficiently cleaved the fusion proteins bound to the four affinity matrices. By combination with appropriate affinity handles, the cognate TEVp5M mediating tag removal enabled purification and cleavage of the fusion proteins, removal of the protease, and separation of the target proteins from the affinity resin to be accomplished in one step.

NAT10 regulates p53 activation through acetylating p53 at K120 and ubiquitinating Mdm2.

As a genome guardian, p53 maintains genome stability by arresting cells for damage repair or inducing cell apoptosis to eliminate the damaged cells in stress response. Several nucleolar proteins stabilize p53 by interfering Mdm2–p53 interaction upon cellular stress, while other mechanisms by which nucleolar proteins activate p53 remain to be determined. Here, we identify NAT10 as a novel regulator for p53 activation. NAT10 acetylates p53 at K120 and stabilizes p53 by counteracting Mdm2 action. In addition, NAT10 promotes Mdm2 degradation with its intrinsic E3 ligase activity. After DNA damage, NAT10 translocates to nucleoplasm and activates p53‐mediated cell cycle control and apoptosis. Finally, NAT10 inhibits cell proliferation and expression of NAT10 decreases in human colorectal carcinomas. Thus, our data demonstrate that NAT10 plays a critical role in p53 activation via acetylating p53 and counteracting Mdm2 action, providing a novel pathway by which nucleolar protein activates p53 as a cellular stress sensor.

Inhibition of cell proliferation, migration and invasion by a glioma-targeted fusion protein combining the p53 C terminus and MDM2-binding domain.

The aim of this study was to develop multifunctional fusion proteins for targeting and delivering therapy elements into glioma cells. Multifunctional fusion proteins were expressed in Escherichia coli and purified using Ni-NTA resin affinity chromatography. Human glioma cells and primary astrocytes were used to analyse their functions. Targeting proteins location to glioma cells was observed by confocal microscopy. Effects of cell viability and proliferation were evaluated using the Cell Counting Kit 8 and colony formation assays. Glioma cell migration and invasion were assessed using transwell assays, and apoptosis was analysed by flow cytometry. In addition, changes in expression of proteins related to the cell cycle and apoptosis were determined by Western blotting. The protein with highest bioactivity was GL1-riHA2-p53c+m-TAT (GHPc+mT), which combines glioma-targeting peptide GL1 (G), and C terminus (Pc) and mouse double minute domains (Pm) of p53, with the destabilizing lipid membrane peptide riHA2 (H) and cell-penetrating peptide TAT (T). The purified fusion protein was stable in cell culture medium and specifically targeted, and was internalized by, epidermal growth factor receptor (EGFR)-overexpressing glioma cells (U87ΔEGFR). It inhibited cell proliferation, migration and invasion, while flow cytometric analysis showed increased apoptosis. In addition, GHPc+mT caused significant changes in expression of proteins related to the cell cycle and apoptosis. GHPc+mT is a multifunctional protein combining targeting, inhibition of glioma cell proliferation and induction of apoptosis, providing some potential to be developed into an effective protein drug delivery system for glioma therapy.

Routes to improve binding capacities of affinity resins demonstrated for Protein A chromatography.

Protein A chromatography is a well-established platform in downstream purification of monoclonal antibodies. Dynamic binding capacities are continuously increasing with almost every newly launched Protein A resin. Nevertheless, binding capacities of affinity chromatography resins cannot compete with binding capacities obtained with modern ion exchange media. Capacities of affinity resins are roughly 50% lower. High binding capacities of ion exchange media are supported by spacer technologies. In this article, we review existing spacer technologies of affinity chromatography resins. A yet known effective approach to increase the dynamic binding capacity of Protein A resins is oligomerization of the particular Protein A motifs. This resembles the tentacle technology used in ion exchange chromatography. Dynamic binding capacities of a hexameric ligand are roughly twice as high compared to capacities obtained with a tetrameric ligand. Further capacity increases up to 130mg/ml can be realized with the hexamer ligand, if the sodium phosphate buffer concentration is increased from 20 to 100mM. Equilibrium isotherms revealed a BET shape for the hexamer ligand at monoclonal antibody liquid phase concentrations higher than 9mg/ml. The apparent multilayer formation may be due to hydrophobic forces. Other quality attributes such as recovery, aggregate content, and overall purity of the captured monoclonal antibody are not affected.

Production of Recombinant Cholera Toxin B Subunit in Nicotiana benthamiana Using GENEWARE® Tobacco Mosaic Virus Vector.

Here, we describe a method to produce a recombinant cholera toxin B subunit in Nicotiana benthamiana plants (CTBp) using the GENEWARE(®) tobacco mosaic virus vector system. Infectious transcripts of the vector RNA are generated in vitro and inoculated on N. benthamiana seedlings. After 11 days, CTBp is extracted in a simple tris buffer at room temperature. No protease inhibitor is required. The leaf homogenate is treated with mild heat and a pH shift to selectively precipitate host-derived proteins. CTBp is purified to >95 % homogeneity by two-step chromatography using immobilized metal affinity and ceramic hydroxyapatite resins. This procedure yields on average 400 mg of low-endotoxin CTBp from 1 kg of fresh leaf material.

Evaluation of employing poly-lysine tags versus poly-histidine tags for purification and characterization of recombinant copper-binding proteins

Quantitative characterization of metalloproteins at molecular and atomic levels generally requires tens of milligrams of highly purified samples, a situation frequently challenged by problems in generating unmodified native forms. A variety of affinity tags, such as the popular poly-histidine tag, have been developed to facilitate purification but they generally rely on expensive affinity resins and their presence may interfere with protein characterization. This paper documents that addition of a poly-lysine tag to the C-terminus enables, for the copper-binding proteins examined, ready purification in large scale via cost-effective cation-exchange chromatography. The tag may be removed readily by the enzyme carboxypeptidase B to generate the native protein with no extra residues. However, this cleavage step is normally not necessary since the poly-lysine tag is shown to have no detectable affinity for either Cu(I) or Cu(II) and imposes no interference to the copper binding properties of the target proteins. In contrast, the poly-histidine tag possesses a sub-picomolar affinity for Cu(I) and -nanomolar affinity for Cu(II) and may need to be removed for reliable characterization of the target proteins. These conclusions may be extended to the study of other metallo-proteins and metallo-enzymes.

Molecular Identification of Potential Probiotic Lactic Acid Bacteria Strains Isolated from Egyptian Traditional Fermented Dairy Products

In order to obtain oligonucleotide aptamers which can specifically recognize the cytotoxin ricin protein,we designed and synthesized an 87 nucleotides ssDNA pool containing 40 random nucleotides flanked by invariant binding regions for PCR primers,which could be used to screen out a specific aptamer for ricin by SELEX (systematic evolution of ligands by exponential enrichment).After several rounds selection,the 38.5% binding ratio of the ssDNA pool bound to ricin protein can be obtained.The results show that the high efficiency of enrichment has been observed by using affinity resin as separation medium.

A laboratory exercise to illustrate protein-membrane interactions.

The laboratory protocol presented here takes about 3 hours to perform and investigates protein and lipid interactions. Students first purify His6 -tagged human apolipoprotein A-I (apoA-I) with Ni-NTA affinity resin in a simple batch protocol and prepare multilamellar vesicles (MLV) from pre-dried phospholipid films. When apoA-I is added to the MLV, much smaller protein/lipid nanodisc complexes are formed in some instances. Nanodisc formation can be monitored by a decrease in light-scattering intensity at 340 nm using a simple spectrophotometer. Students will observe nanodisc formation with MLV formed from the anionic phospholipid dimyristoylphosphatidyl glycerol, which pack poorly into lipid bilayers, but not with MLV formed from the zwitterionic phospholipid dimyristoyl phosphatidylcholine, which form stable bilayers. This laboratory exercise is accompanied by questions and exercises that enable students a deeper of the dimensions of apoA-I and nanodiscs as well as the biological relevance of nanodisc formation in the process of reverse cholesterol transport.

The elution of certain protein affinity tags with millimolar concentrations of diclofenac

Diclofenac (2-[(2, 6-dichlorophenyl)amino] benzeneacetic acid) is a sparingly soluble, nonsteroidal anti-inflammatory drug therapeutically acting at low micromolar concentrations. In pH range from 8 to 11, its aqueous solubility can be increased up to 200 times by the presence of counter ions such as sodium. Our protein interaction studies revealed that a millimolar concentration of sodium diclofenac is able to elute glutathione S-transferase (GST), cellulose binding protein (CBD), and maltose binding protein (MBP) but not histidine-tagged or PDZ-tagged proteins from their affinity resins. The elution efficiency of diclofenac is comparable with the eluting agents normally used at similar concentrations. Native gel electrophoresis of sodium diclofenac-treated proteins showed that the interaction is non-covalent and non-denaturing. These results suggest that sodium diclofenac, in addition to its pharmaceutical applications, can also be exploited as a lead for the development of new proteomics reagents.

Molecular Mechanisms and Kinetic Effects of FXYD1 and Phosphomimetic Mutants on Purified Human Na,K-ATPase

Phospholemman (FXYD1) is a single-transmembrane protein regulator of Na,K-ATPase, expressed strongly in heart, skeletal muscle, and brain and phosphorylated by protein kinases A and C at Ser-68 and Ser-63, respectively. Binding of FXYD1 reduces Na,K-ATPase activity, and phosphorylation at Ser-68 or Ser-63 relieves the inhibition. Despite the accumulated information on physiological effects, whole cell studies provide only limited information on molecular mechanisms. As a complementary approach, we utilized purified human Na,K-ATPase (α1β1 and α2β1) reconstituted with FXYD1 or mutants S63E, S68E, and S63E,S68E that mimic phosphorylation at Ser-63 and Ser-68. Compared with control α1β1, FXYD1 reduces Vmax and turnover rate and raises K0.5Na. The phosphomimetic mutants reverse these effects and reduce K0.5Na below control K0.5Na. Effects on α2β1 are similar but smaller. Experiments in proteoliposomes reconstituted with α1β1 show analogous effects of FXYD1 on K0.5Na, which are abolished by phosphomimetic mutants and also by increasing mole fractions of DOPS in the proteoliposomes. Stopped-flow experiments using the dye RH421 show that FXYD1 slows the conformational transition E2(2K)ATP → E1(3Na)ATP but does not affect 3NaE1P → E2P3Na. This regulatory effect is explained simply by molecular modeling, which indicates that a cytoplasmic helix (residues 60-70) docks between the αN and αP domains in the E2 conformation, but docking is weaker in E1 (also for phosphomimetic mutants). Taken together with previous work showing that FXYD1 also raises binding affinity for the Na(+)-selective site III, these results provide a rather comprehensive picture of the regulatory mechanism of FXYD1 that complements the physiological studies.

27 High definition lipoproteomics reveal dysregulated redox proteins in coronary artery disease

Background Lipoproteins have an integral role in the pathogenesis of CAD. Quantitative proteomics is evolving to become an indispensable tool in the era of precision medicine. Using an unbiased lipoproteomic discovery workflow we sought to investigate the lipoproteomic differences between stable CAD patients and controls subjects and to reveal novel pathways/mechanisms that underpin this common disease. Methods Male patients with stable CAD (n = 49, mean ± SD; age 64 yrs ± 8.69) and without CAD (n = 17; mean age 74 yrs ± 6.21) were recruited into this single centre prospective cohort study. Plasma was co-incubated with a lipophilic affinity resin. Isolated lipoproteins were reduced, alkylated, digested and analysed using label-free high definition ion mobility enabled mass spectrometry. Raw data was analysed using Progenesis QI software with a stringent false discovery rate of 1%. Results 202 proteins showed significant differential expression between the CAD patients and the control subjects (P Conclusion Patients with CAD have a distinct pathognomonic lipoproteomic cargo. The measurement of certain lipoprotein associated proteins may assist in the early identification of such patients before symptom onset.

Evaluation of Kinase Activity Profiling Using Chemical Proteomics.

Protein kinases are important mediators of intracellular signaling and are reversibly activated by phosphorylation. Immobilized kinase inhibitors can be used to enrich these often low-abundance proteins, to identify targets of kinase inhibitors, or to probe their selectivity. It has been suggested that the binding of kinases to affinity beads reflects a kinase's activation status, a concept that is under considerable debate. To assess the merits of the idea, we performed a series of experiments including quantitative phosphoproteomics and purification of kinases by single or mixed affinity matrices from signaling activated or resting cancer cells. The data show that mixed affinity beads largely bind kinases independent of their activation status, and experiments using individual immobilized kinase inhibitors show mixed results in terms of preference for binding the active or inactive conformation. Taken together, activity- or conformation-dependent binding to such affinity resins depends (i) on the kinase, (ii) on the affinity probe, and (iii) on the activation status of the lysate or cell. As a result, great caution should be exercised when inferring kinase activity from such binding data. The results also suggest that assaying kinase activity using binding data is restricted to a limited number of well-chosen cases.

Chromium(III) Binding Phage Screening for the Selective Adsorption of Cr(III) and Chromium Speciation.

The screening of suitable sorption medium is the key for highly selective solid phase extraction (SPE) of heavy metals. Herein, we demonstrate a universal protocol for producing selective SPE adsorbent through an evolutional approach based on phage display peptide library. By choosing chromium(III) as the model target, immobilized Cr(III) resins are first prepared using Ni-NTA affinity resins for the interaction with NEB heptapeptide phage library. After three rounds of positive biopanning against target Cr(III) and negative biopanning against foreign metal species, Cr(III) binding phages with high selectivity are obtained. The binding affinity and selectivity are further assessed with ELISA. The phages bearing peptide (YKASLIT) is finally chosen and immobilized on cytopore beads for Cr(III) preconcentration. The retained Cr(III) is efficiently recovered by 0.10 mol L(-1) HNO3 and quantified with ICP-MS. By loading 4000 μL of sample solution at pH 7.0 for 2 h and stripping with 400 μL of 0.10 mol L(-1) HNO3, a linear range of 0.05-0.50 μg L(-1) is achieved along with an enrichment factor of 7.1. The limit of detection is derived to be 15 ng L(-1) (3σ, n = 7) with a RSD of 3.6% (0.25 μg L(-1), n = 7). The procedure is validated by analyzing chromium content in a certified reference material GBW08608 (simulate water). In addition, chromium speciation in real water samples is demonstrated. Cr(VI) is first converted into Cr(III), and the latter subjected to the sorption onto the Cr(III) binding phage, followed by elution and quantification of the total chromium amount, and finally speciation is achieved by difference.

Development, upscaling and validation of the purification process for human-cl rhFVIII (Nuwiq®), a new generation recombinant factor VIII produced in a human cell-line

IntroductionHuman-cl rhFVIII (Nuwiq®), a new generation recombinant factor VIII (rFVIII), is the first rFVIII produced in a human cell-line approved by the European Medicines Agency. AimsTo describe the development, upscaling and process validation for industrial-scale human-cl rhFVIII purification. Methods and resultsThe purification process involves one centrifugation, two filtration, five chromatography columns and two dedicated pathogen clearance steps (solvent/detergent treatment and 20nm nanofiltration). The key purification step uses an affinity resin (VIIISelect) with high specificity for FVIII, removing essentially all host-cell proteins with >80% product recovery. The production-scale multi-step purification process efficiently removes process- and product-related impurities and results in a high-purity rhFVIII product, with an overall yield of ∼50%. Specific activity of the final product was >9000IU/mg, and the ratio between active FVIII and total FVIII protein present was >0.9. The entire production process is free of animal-derived products. Leaching of potential harmful compounds from chromatography resins and all pathogens tested were below the limit of quantification in the final product. ConclusionsHuman-cl rhFVIII can be produced at 500L bioreactor scale, maintaining high purity and recoveries. The innovative purification process ensures a high-purity and high-quality human-cl rhFVIII product with a high pathogen safety margin.