Anion-exchange Chromatography Steps Research Articles

A manganese catalase from Thermomicrobium roseum with peroxidase and catecholase activity.

An enzyme with catechol oxidase activity was identified in Thermomicrobium roseum extracts via solution assays and activity-stained SDS-PAGE. Yet, the genome of T. roseum does not harbor a catecholase gene. The enzyme was purified with two anion exchange chromatography steps and ultimately identified to be a manganese catalase with additional peroxidase and catecholase activity. Catalase activity (6280±430IU/mg) clearly dominated over pyrogallol peroxidase (231±53IU/mg) and catecholase (3.07±0.56IU/mg) activity as determined at 70°C. Most enzyme kinetic properties were comparable to previously characterized manganese catalase enzymes. Catalase activity was highest at alkaline pH values and showed inhibition by excess substrate and chloride. The apparent K m and k cat values were 20mM and 2.02×104s-1 subunit-1 at 25°C and pH 7.0.

Expression, purification and crystallization of the (3R)-hydroxyacyl-ACP dehydratase HadAB complex from Mycobacterium tuberculosis

The (3R)-hydroxyacyl-ACP dehydratase HadAB, involved in the biosynthetic pathway for mycolic acid (MA) of Mycobacterium tuberculosis, catalyzes the third step in the fatty acid (FA) elongation cycle, which is an ideal and actual target for anti-tubercular agent. Though HadAB is predicted to be a member of the hotdog superfamily, it shares no sequence identity with typical hotdog fold isoenzyme FabZ. To characterize the significance of HadAB from the perspective of structural biology, large amount of pure HadAB complex is required for biochemical characterization and crystallization. Here, we used a unique expression and purification method. HadA and HadB were cloned separately and co-expressed in Escherichia coli. After GST affinity chromatography, two steps of anion exchange chromatography and gel filtration, the purity of the protein as estimated by SDS–PAGE was >95%. Using hanging-drop vapor-diffusion method, crystals were obtained and diffracted X-rays to 1.75Å resolution. The crystal belongs to space group P41212, with unit-cell parameters a=b=82.0Å, c=139.8Å, α=β=γ=90.0°.

The ascomycete Xylaria polymorpha produces an acetyl esterase that solubilises beech wood material to release water-soluble lignin fragments

Three ascomycetous soft-rot fungi belonging to the family Xylariaceae were found to produce high levels of a p-nitrophenyl acetate-hydrolyzing activity during growth on lignocellulosic materials, i.e., wheat straw and beech wood chips. Accordingly, Kretzschmaria deusta, Morchella elata, Xylaria polymorpha were seemingly most noteworthy acetyl esterase producers and, of which, X. polymorpha (strain A35) was chosen for further studies. Induction study indicated that raw carbohydrate sources, such as beech wood, rape straw, birch wood, and wheat straw, were extremely important for acetyl esterase production. Acetyl esterase of X. polymorpha was produced in solid-state culture on wheat straw and purified by different steps of anion-exchange and size-exclusion chromatography. This purified enzyme (M W = 44 kDa and pI values of 3.5–3.6) exhibited the capability to solubilise in vitro beech wood to release water-soluble lignin fragments with molecular masses of 1–3 kDa as analyzed by high performance size exclusion chromatography.

Mitigation of chromatography adsorbent lot performance variability through control of buffer solution design space

The separation of undesired product-related impurities often poses a challenge in the purification of protein therapeutic species. Product-related impurity species, which may consist of undesirable isoforms, aggregated, or misfolded variants of the desired monomeric form of the product, can be challenging to remove using preparatory scale chromatographic techniques. When using anion exchange chromatography to remove undesirable product-related impurities, the separation can be highly sensitive to relatively small changes in the chromatography operating conditions, including changes to buffer solution pH, buffer solution conductivity protein loading, and operating temperature. When performing difficult separations, slight changes to the chemical and physical properties of the anion exchange adsorbent lot may also impact the separation profile. Such lot-to-lot variability may not be readily measurable by the adsorbent manufacturer, since variability can be highly dependent on a specific protein separation. Consequently, manufacturers of chromatographic adsorbents may not be able to control adsorbent lot to lot variability tightly enough to prevent differences from occurring when performing difficult product-related separations at the preparatory scale. In such cases, it is desirable to design a chromatography step with a control strategy which accounts for adsorbent lot to lot variability in the separation performance. In order to avoid the undesired changes to process consistency and product quality, a proper adjustment of the column operating conditions can be implemented, based on the performance of each adsorbent lot or lot mixture. In this work, we describe how the adjustment of the column buffer solution composition can be used as a design space based-control strategy used to ensure consistent process performance and product quality are achieved for an anion exchange chromatography step susceptible to adsorbent lot to lot performance variability. In addition, a “use test” is described that can be employed to determine the optimal buffer solution compositions for different anion exchange adsorbent lots based on the retention volume of the therapeutic protein during a gradient elution.

Development of a Human IgG4 Bispecific Antibody for Dual Targeting of Interleukin-4 (IL-4) and Interleukin-13 (IL-13) Cytokines

Human bispecific antibodies have great potential for the treatment of human diseases. Although human IgG1 bispecific antibodies have been generated, few attempts have been reported in the scientific literature that extend bispecific antibodies to other human antibody isotypes. In this paper, we report our work expanding the knobs-into-holes bispecific antibody technology to the human IgG4 isotype. We apply this approach to generate a bispecific antibody that targets IL-4 and IL-13, two cytokines that play roles in type 2 inflammation. We show that IgG4 bispecific antibodies can be generated in large quantities with equivalent efficiency and quality and have comparable pharmacokinetic properties and lung partitioning, compared with the IgG1 isotype. This work broadens the range of published therapeutic bispecific antibodies with natural surface architecture and provides additional options for the generation of bispecific antibodies with differing effector functions through the use of different antibody isotypes.

Purification of recombinant vaccinia virus-expressed monomeric HIV-1 gp120 to apparent homogeneity

Vaccinia virus (VV) has been used to express a variety of heterologous proteins, including HIV envelope (Env) glycoproteins. The Env protein is synthesized as a precursor molecule, gp160, which is cleaved into the surface antigen gp120 and the transmembrane protein gp41. Even though production of gp160 by the VV expression system has been described, its use for gp120 production is not well documented. Here we report a new procedure for the purification of gp120 from serum-containing culture supernatant of VV-infected cells. The gp120 protein was enriched to a purity better than 60% on a snowdrop (Galanthus nivalis) lectin affinity column in the presence of 0.25% zwitterionic detergent Empigen BB. After additional DEAE anion exchange and Superdex size exclusion chromatography steps, the gp120 monomer was purified free of contamination as determined by SDS–PAGE. The retention of structural integrity was confirmed by determining the affinity constant of purified gp120s to soluble CD4 and a monoclonal antibody IgG1b12, using surface plasmon resonance analysis. The purification procedure is robust and reproducible, and may find general use for glycoprotein purifications from sources where the presence of serum is desirable.

Biochemical Characteristics of New Delhi Metallo-β-Lactamase-1 Show Unexpected Difference to Other MBLs

New Delhi metallo-β-lactamase (NDM-1) is a new metallo-β-lactamase (MBL) that has recently emerged as a global threat because it confers bacteria with resistance to almost all clinically used β-lactam antibiotics. To determine the molecular basis of this threat, NDM-1 was purified from Escherichia coli TransB (DE3) carrying cloned blaNDM-1 gene by an anion-exchange chromatography step followed by a gel permeation chromatography step. The purified enzyme was stable even in extremely alkaline buffer (pH 11) and reached its highest activity at a low temperature (15°C), which was different from other MBLs. The 50% inhibition concentration of EDTA against NDM-1 was 412 nM, which showed that NDM-1 was more susceptible to EDTA than other MBLs. The effects of zinc on NDM-1 differed between cephem and carbapenem complexes, but inhibition at high Zn2+ concentration was observed for all of tested β-lactam compounds.

Purification and use of E. coli peptide deformylase for peptide deprotection in chemoenzymatic peptide synthesis

Peptide deformylases (PDFs) catalyze the removal of the formyl group from the N-terminal methionine residue in nascent polypeptide chains in prokaryotes. Its deformylation activity makes PDF an attractive candidate for the biocatalytic deprotection of formylated peptides that are used in chemoenzymatic peptide synthesis. For this application it is essential to use PDF preparations that are free of contamination by peptidases that can cleave internal peptide bonds. Therefore, different purification methods were attempted and an industrially applicable purification procedure was developed based on a single anion-exchange chromatography step of an engineered PDF variant that was equipped with an anionic octaglutamate tag. The deformylation activity and stability of the engineered enzyme were similar to those of the wild-type PDF. This purification method furnished a PDF preparation with a 1500-fold decreased level of contamination by amidases and peptidases as compared to cell-free extract. It was shown that the enzyme could be used for deprotection of a formylated dipeptide that was prepared by thermolysin-mediated coupling.

Recombinant expression, purification and characterisation of the native glutamate racemase from Lactobacillus plantarum NC8

Glutamic acid racemases (MurI, E.C. 5.1.1.3) catalyse the racemisation of l- and d-glutamic acid. MurIs are essential enzymes for bacterial cell wall synthesis, which requires d-glutamic acid as an indispensable building block. Therefore these enzymes are suitable targets for antimicrobial drugs as well as for the potential design of auxotrophic selection markers. A high expression system in Escherichia coli BL21 (DE3) was constructed to produce and characterise the biochemical properties of the MurI from Lactobacillus plantarum NC8. In a 4-L-bioreactor cultivation, 3266nkatd-Glu/mgprotein of specific enzyme activity was produced. The recombinant, tag-free Murl was purified by an innovative affinity chromatography method using l-glutamic acid as the relevant docking group, followed by an anion exchange chromatography step (purification factor 9.2, yield 11%). This two-step purification strategy resulted in a Murl sample with a specific activity of 34,060nkatd-Glu/mgprotein, comprising a single protein band in SDS–PAGE. The purified Murl possessed an assay temperature optimum of 50°C, but it was not stable at this temperature. The half-lives of the purified Murl were 162h at 20°C and only 1.9h at 40°C. The Murl activity was maximum between pH 7 and 10, resulting in a maximal half-life of 287h at pH 7. Only d- and l-glutamic acid were recognised as substrates for the Murl with similar Kcat/KM ratios of 3.6s−1/mM for each enantiomer.

Expression and purification of chimeric peptide comprising EGFR B-cell epitope and measles virus fusion protein T-cell epitope in Escherichia coli

Chimeric peptide MVF-EGFR237–267, comprising a B-cell epitope from the dimerization interface of human epidermal growth factor receptor (EGFR) and a promiscuous T-cell epitope from measles virus fusion protein (MVF), is a promising candidate antigen peptide for therapeutic vaccine. To establish a high-efficiency preparation process of this small peptide, the coding sequence was cloned into pET-21b and pET-32a respectively, to be expressed alone or in the form of fusion protein with thioredoxin (Trx) and His6-tag in Escherichia coli BL21 (DE3). The chimeric peptide failed to be expressed alone, but over-expressed in the fusion form, which presented as soluble protein and took up more than 30% of total proteins of host cells. The fusion protein was seriously degraded during the cell disruption, in which endogenous metalloproteinase played a key role. Degradation of target peptide was inhibited by combined application of EDTA in the cell disruption buffer and a step of Source 30Q anion exchange chromatography (AEC) before metal-chelating chromatography (MCAC) for purifying His6-tagged fusion protein. The chimeric peptide was recovered from the purified fusion protein by enterokinase digestion at a yield of 3.0mg/L bacteria culture with a purity of more than 95%. Immunogenicity analysis showed that the recombinant chimeric peptide was able to arouse more than 1×104 titers of specific antibody in BALB/c mice. Present work laid a solid foundation for the development of therapeutic peptide vaccine targeting EGFR dimerization and provided a convenient and low-cost preparation method for small peptides.

Automated multi-step purification protocol for Angiotensin-I-Converting-Enzyme (ACE)

Highly purified proteins are essential for the investigation of the functional and biochemical properties of proteins. The purification of a protein requires several steps, which are often time-consuming. In our study, the Angiotensin-I-Converting-Enzyme (ACE; EC 3.4.15.1) was solubilised from pig lung without additional detergents, which are commonly used, under mild alkaline conditions in a Tris–HCl buffer (50mM, pH 9.0) for 48h. An automation of the ACE purification was performed using a multi-step protocol in less than 8h, resulting in a purified protein with a specific activity of 37Umg−1 (purification factor 308) and a yield of 23.6%. The automated ACE purification used an ordinary fast-protein-liquid-chromatography (FPLC) system equipped with two additional switching valves. These switching valves were needed for the buffer stream inversion and for the connection of the Superloop™ used for the protein parking. Automated ACE purification was performed using four combined chromatography steps, including two desalting procedures. The purification methods contained two hydrophobic interaction chromatography steps, a Cibacron 3FG-A chromatography step and a strong anion exchange chromatography step. The purified ACE was characterised by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and native-PAGE. The estimated monomer size of the purified glycosylated ACE was determined to be ∼175kDa by SDS-PAGE, with the dimeric form at ∼330kDa as characterised by a native PAGE using a novel activity staining protocol. For the activity staining, the tripeptide l-Phe-Gly-Gly was used as the substrate. The ACE cleaved the dipeptide Gly-Gly, releasing the l-Phe to be oxidised with l-amino acid oxidase. Combined with peroxidase and o-dianisidine, the generated H2O2 stained a brown coloured band. This automated purification protocol can be easily adapted to be used with other protein purification tasks.

A 2,3‐butanediol dehydrogenase from Paenibacillus polymyxaZJ‐9 for mainly producing R,R‐2,3‐butanediol: Purification, characterization and cloning

A 2,3-butanediol dehydrogenase (BDH) from Paenibacillus polymyxa ZJ-9 was purified to homogeneity via fractional ammonium sulfate precipitation, followed by two steps of anion-exchange chromatography using DEAE-Sepharose and Source 15Q, obtaining a 35-fold increase in specific activity and 34.9% yield. The molecular weights of the purified BDH subunit and holoenzyme were 44.5 and 90.0 kDa, respectively, as detected via SDS-PAGE and gel filtration chromatography. These results were significantly different from those of other reported BDHs. Substrate specificity experiments showed that the enzyme could function preferentially as a reductase rather than as a dehydrogenase, and was mainly responsible for the reduction of R-acetoin to R,R-2,3-butanediol. Gene cloning, sequencing, and expression experiments further demonstrate that this enzyme was a new type of BDH.

Specialized Hsp70 Chaperone (HscA) Binds Preferentially to the Disordered Form, whereas J-protein (HscB) Binds Preferentially to the Structured Form of the Iron-Sulfur Cluster Scaffold Protein (IscU)

The Escherichia coli protein IscU serves as the scaffold for Fe-S cluster assembly and the vehicle for Fe-S cluster transfer to acceptor proteins, such as apoferredoxin. IscU populates two conformational states in solution, a structured conformation (S) that resembles the conformation of the holoprotein IscU-[2Fe-2S] and a dynamically disordered conformation (D) that does not bind metal ions. NMR spectroscopic results presented here show that the specialized Hsp70 chaperone (HscA), alone or as the HscA-ADP complex, preferentially binds to and stabilizes the D-state of IscU. IscU is released when HscA binds ATP. By contrast, the J-protein HscB binds preferentially to the S-state of IscU. Consistent with these findings, we propose a mechanism in which cluster transfer is coupled to hydrolysis of ATP bound to HscA, conversion of IscU to the D-state, and release of HscB.

Chromatography Purification of Canine Adenoviral Vectors

Canine adenovirus vectors (CAV2) are currently being evaluated for gene therapy, oncolytic virotherapy, and as vectors for recombinant vaccines. Despite the need for increasing volumes of purified CAV2 preparations for preclinical and clinical testing, their purification still relies on the use of conventional, scale-limited CsCl ultracentrifugation techniques. A complete downstream processing strategy for CAV2 vectors based on membrane filtration and chromatography is reported here. Microfiltration and ultra/diafiltration are selected for clarification and concentration of crude viral stocks containing both intracellular and extracellular CAV2 particles. A DNase digestion step is introduced between ultrafiltration and diafiltration operations. At these early stages, concentration of vector stocks with good recovery of viral particles (above 80%) and removal of a substantial amount of protein and nucleic acid contaminants is achieved. The ability of various chromatography techniques to isolate CAV2 particles was evaluated. Hydrophobic interaction chromatography using a Fractogel propyl tentacle resin was selected as a first chromatography step, because it allows removal of the bulk of contaminating proteins with high CAV2 yields (88%). An anion-exchange chromatography step using monolithic supports is further introduced to remove the remaining contaminants with good recovery of CAV2 particles (58-69%). The main CAV2 viral structural components are visualized in purified preparations by electrophoresis analyses. Purified vector stocks contained intact icosahedral viral particles, low contamination with empty viral capsids (10%), and an acceptable total-to-infectious particle ratio (below 30). The downstream processing strategy that was developed allows preparation of large volumes of high-quality CAV2 stocks.

The Wood Rot Ascomycete Xylaria polymorpha Produces a Novel GH78 Glycoside Hydrolase That Exhibits α- l -Rhamnosidase and Feruloyl Esterase Activities and Releases Hydroxycinnamic Acids from Lignocelluloses

Soft rot (type II) fungi belonging to the family Xylariaceae are known to substantially degrade hardwood by means of their poorly understood lignocellulolytic system, which comprises various hydrolases, including feruloyl esterases and laccase. In the present study, several members of the Xylariaceae were found to exhibit high feruloyl esterase activity during growth on lignocellulosic materials such as wheat straw (up to 1,675 mU g(-1)) or beech wood (up to 80 mU g(-1)). Following the ester-cleaving activity toward methyl ferulate, a hydrolase of Xylaria polymorpha was produced in solid-state culture on wheat straw and purified by different steps of anion-exchange and size-exclusion chromatography to apparent homogeneity (specific activity, 2.2 U mg(-1)). The peptide sequence of the purified protein deduced from the gene sequence and verified by de novo peptide sequencing shows high similarity to putative α-L-rhamnosidase sequences belonging to the glycoside hydrolase family 78 (GH78; classified under EC 3.2.1.40). The purified enzyme (98 kDa by SDS-PAGE, 103 kDa by size-exclusion chromatography; pI 3.7) converted diverse glycosides (e.g., α-L-rhamnopyranoside and α-L-arabinofuranoside) but also natural and synthetic esters (e.g., chlorogenic acid, hydroxycinnamic acid glycoside esters, veratric acid esters, or p-nitrophenyl acetate) and released free hydroxycinnamic acids (ferulic and coumaric acid) from arabinoxylan and milled wheat straw. These catalytic properties strongly suggest that X. polymorpha GH78 is a multifunctional enzyme. It is the first fungal enzyme that combines glycosyl hydrolase with esterase activities and may help this soft rot fungus to degrade lignocelluloses.

Purification of norovirus-like particles (VLPs) by ion exchange chromatography

Recombinant expression of the norovirus capsid protein VP1 leads to self-assembly of non-infectious virus-like particles (VLPs), which are recognized as promising vaccine candidates against norovirus infections. To overcome the scalability issues connected to the ultracentrifugation-based purification strategies used in previous studies, an anion exchange-based purification method for norovirus VLPs was developed in this study. The method consists of precipitation by polyethylene glycol (PEG) and a single anion exchange chromatography step for purifying baculovirus-expressed GII.4 norovirus VLPs, which can be performed within one day. High product purity was obtained using chromatography. The purified material also contained fully assembled monodispersed VLPs, which were recognized by human sera containing polyclonal antibodies against norovirus GII.4.

Highly Purified Inner-Arm Dynein E from Chlamydomonas Reinhardtii Studied with in vitro Motility Assay

Green alga Chlamydomonas reinhardtii has two flagella at the front of the cell and beats them to swim. A flagellium is a biomolecular machine that consists of various proteins in a highly regulated manner. It is considered that an alignment of the various species of dynein proteins, which generate power using energy released upon ATP hydrolysis, plays important roles in the flagellum beating. Recently, great progress has been made in understanding of construction of a Chlamydomonas flagellum by the use of cryo-electron microscopic tomography. Dyneins are arranged in 96-nm periodic manner for a direction of a longitudinal axis of a flagellum. 8 species of dyneins are called as “inner-arm dyneins”. In 96-nm period, they make specific 4 pairs and are arranged in equally-spaced 4 places. Making use of in vitro motility assay, we can evaluate activities of dyneins as “microtubule sliding”. Dynein “c” slides microtubules relatively fast among the 8 inner-arms, and is well-studied. It has shown that dynein c pairs with dynein “e” in a flagellium. However, properties of dynein e are not known much. One reason for the difficulty is that anion exchange chromatography, a major purification method for inner arm dyneins, cannot separate dynein e and dynein d well. We obtained highly purified dynein e and c using two steps of anion exchange chromatography. We will present properties of dynein e and of a mixture of dynein e and c.

Separation of supercoiled from open circular forms of plasmid DNA, and biological activity detection

To establish a cost-effective purification process for the large-scale production of plasmid DNA for gene therapy and DNA vaccination, a single anion-exchange chromatography (AEC) step was employed to purify supercoiled plasmid DNA (sc pDNA) from other isoforms and Escherichia coli impurities present in a clarified lysate. Two different size and conformation plasmids were used as model targets, and showed similar elution behavior in this chromatographic operation, in which sc pDNA was effectively separated from open circle plasmid DNA (oc pDNA) in a salt gradient. The process delivered high-purity pDNA of homogeneity of 95±1.1% and almost undetectable levels of endotoxins, genomic DNA, RNA and protein, at a yield of 65±8%. Furthermore, the transfection efficiency (29±0.4%) was significantly higher than that (20±0.1%) of a pDNA control. The present study confirms the possibility of using a single AEC step to purify sc pDNA from other isoforms and host contaminants present in a clarified E. coli lysate.

Highly efficient production of phosphorylated hepatitis B core particles in yeast Pichia pastoris

Virus-like particles (VLPs) of the recombinant hepatitis B virus (HBV) core protein (HBc) are routinely used in HBV diagnostics worldwide and are of potential interest as carriers of foreign peptides (e.g., immunological epitopes and targeting addresses, and/or as vessels for packaged diagnostic and therapeutic nanomaterials). Despite numerous reports exploiting different expression systems, a rapid and comprehensive large-scale methodology for purification of HBc VLPs from yeast is still lacking. Here, we present a convenient protocol for highly efficient production and rapid purification of endotoxin-free ayw subtype HBc VLPs from the methylotrophic yeast Pichia pastoris. The HBc gene expression cassette along with the geneticin resistance gene was transferred to the P. pastoris genome via homologous recombination. A producer clone was selected among 2000 transformants for the optimal synthesis of the target protein. Fermentation conditions were established ensuring biomass accumulation of 163 g/L. A simple combination of pH/heat and salt treatment followed by a single anion-exchange chromatography step resulted in a more than 90% pure preparation of HBc VLPs, with a yield of about 3.0 mg per 1 g of wet cells. Purification is performed within a day and may be easily scaled up if necessary. The quality of HBc VLPs was verified by electron microscopy. Mass spectrometry analysis and direct polyacrylamide gel staining revealed phosphorylation of HBc at at least two sites. To our knowledge, this is the first report of HBc phosphorylation in yeast.

Monoclonal antibody purification using cationic polyelectrolytes: An alternative to column chromatography

The potential of cationic polyelectrolytes to precipitate host cell and process related impurities was investigated, to replace one or more chromatography steps in monoclonal antibody purification. The impact of antibody isoelectric point, solution properties (pH and ionic strength), and polyelectrolyte properties (structure, molecular weight and pK(a)) on the degree of precipitation was studied. At neutral pH, increasing solution ionic strength impeded the ionic interaction between the polyelectrolyte and impurities, reducing impurity precipitation. Increasing polyelectrolyte molecular weight and pK(a) enabled precipitation of impurities at higher ionic strength. PoIy(arginine) was selected as the preferred polyelectrolyte in unconditioned cell culture fluid. PoIy(arginine) precipitation achieved consistent host cell protein clearance and antibody recovery for multiple antibodies across a wider range of polyelectrolyte concentrations. Poly(arginine) precipitation was evaluated as a flocculant and as a functional replacement for anion exchange chromatography in an antibody purification process. Upstream treatment of cell culture fluid with poly(arginine) resulted in flocculation of solids (cells and cell debris), and antibody recovery and impurity clearance (host cell proteins, DNA and insulin) comparable to the downstream anion exchange chromatography step.