Oscar-Werner Reif

Immobilized metal affinity membrane adsorbers as stationary phases for metal interaction protein separation

Abstract Novel immobilized metal affinity membrane adsorbers (IMA-MA) were studied for potential use as stationary phases for protein separation. Protein adsorption on IMA-MA loaded with Cu(II), Ni(II), ZN(II) and Co(II) ions was compared as a function of the flow-rate and the ionic strength of the elution buffer. To exclude the possibility of mixed-mode interaction in the experiments, the binding of proteins similar in terms of hydrophobicity, isoelectric point, size and mass-to-charge ratio but differing in their number of surface histidine residues was investigated. Matrix-assisted laser desorption/ionization mass spectrometry was used to distinguish between these proteins in the eluted fractions. Salt concentration of at least 0.5 M NACl and flow-rates below 2 ml min−1 were found suitable to ensure an adsorption mechanism based on affinity interaction between the proteins and the chelated metal ions. In an application study, the IMA-MA and conventional chelating Sepharose fast flow columns were compared for the isolation of a recombinant fusion protein (EcoR V), which carried a polyhistidine sequence (HIS6-tag) at the N-terminus.

Lipase of Pseudomonas cepacia for biotechnological purposes: purification, crystallization and characterization.

Commercial lipase (triacylglycerol lipase, EC 3.1.1.3) of Pseudomonas cepacia (Amano) has been purified to homogeneity by a single chromatography on phenyl Sepharose. The eluted lipase crystallized spontaneously at 4 degrees C in the eluent, containing 58-69% 2-propanol. The yield of the lipase was 87-100% and the specific activity during the hydrolysis of triolein 5800 U/mg protein. This protein has a molecular weight of 34.1 kDa as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Its purity was determined by SDS-PAGE and capillary zone electrophoresis to be > or = 99%. Immobilization on Sepharose increased its stability in organic solvents. This lipase of P. cepacia differs from that of other Pseudomonas strains in respect to substrate specificity and during crystallization. It exhibits a high stability in organic solvents and supercritical carbon dioxide.

Innovative Modular Membrane Adsorber System for High-Throughput Downstream Screening for Protein Purification

To develop the most efficient strategy for the purification of proteins, two types of adsorber membrane devices with different functionalities were designed and tested: 8‐strips and single spin columns. The most suitable type of membrane adsorber and the optimal chromatographic loading/elution conditions for several target proteins from different biological matrices could be determined simultaneously in microliter scale. Ion exchange (IEX), metal chelate (MC), and Concanavalin A (Con A) modified membrane types were tested in the devices. Bovine serum albumin (BSA) and lysozyme were used as model proteins for investigations of the binding capacity and protein recovery percentage of the 8‐strip anion exchange and the cation exchange membrane. The isolation of His6‐tagged proteins, Bgl‐His and GFP‐His from fermentation broth and lysate, respectively, was performed using an 8‐strip metal chelate affinity membrane loaded with different metal ions. Separation behavior of a ternary protein mixture (BSA, lysozyme, and Bgl‐His) was studied in 8‐strips IEX and metal chelate membrane chromatography. The Con A affinity devices were developed on the basis of metal chelate membrane spin columns loaded with Cu2+ ions and investigated using glucose oxidase (GOD) as model protein. In summary, the advantages of the membrane adsorber technology, such as fast processing and easy scale‐up, were utilized. The devices made it possible to load the membrane directly with preclarified fermentation broth or cell lysate and separate the protein of interest often in a single step.

Controlled mixed-mode interaction chromatography on membrane adsorbers

Abstract Membrane adsorbers (MAs) are used for protein separation in controlled mixed-mode interaction chromatography. The strong anion- and cation-exchange MAs used are made either from a synthetic copolymer or from modified cellulose membranes. The affinity MAs (Cibacron Blue) are also made from the copolymer membranes. Standard protein mixtures, whey proteins, and biotechnological culture supernatants are separated. The influence of the flow-rate, the ratio of cation- and anion-exchange MAs inserted in the stack, and the pattern, i.e. the comparative worth of an alternating vs. a two-consecutive-stacks arrangement, on the separation is investigated. While the flow-rate shows no influence, both the pattern of arrangement and the ratio of the two types of ion exchanger do. Compared to single-mode MA chromatography, a broader range of proteins, e.g. in terms of the isoelectric points, can be separated in a single chromatographic procedure. Whey proteins (β-lactoglobulin, α-lactalbumin, BSA, IgG) are separated at pH 6, using a mixed-mode ion-exchange system. Here however, a two-stack approach is used to allow for module-uncoupling before elution, to prevent IgG and α-lactalbumin from coeluting. By using a mix of anion-exchange and Cibacron Blue affinity MAs, recombinant human antithrombin III (rh-AT III) can be separated in a single run from the major protein impurities present in the fermenter supernatant, namely transferrin and BSA.

Control of the cultivation process of antithrombin III and its characterization by capillary electrophoresis

The production by baby hamster kidney cells of recombinant antithrombin III (r-AT III), the main inhibitor of thrombin, factor Xa and other proteases of the clotting cascade, was monitored by capillary isotachophoresis using mixtures of continuous spacers. The results were compared with those obtained by capillary zone electrophoresis (CZE). The downstream process, which incorporated anion-exchange and heparin affinity chromatography, was monitored by CZE under acidic conditions and voltage ramping. The purified product was characterized by its isoelectric point and molecular mass. Isoelectric points of the three major and three minor isoforms of AT III were evaluated by capillary isoelectric focusing using a pH range of 4-6 and various mobilization procedures. The molecular mass of AT III was investigated by capillary gel electrophoresis (CGE), applying removable dextran gels. Both parameters could be determined within 30 min using only one coated capillary. The results showed an excellent correspondence with those achieved with conventional slab gels. The affinity complex between AT III and thrombin could also be detected by CGE and the heparin dependence of the affinity reaction could be investigated.

Studies of complexes between proteases, substrates and the protease inhibitor α2-macroglobulin using capillary electrophoresis with laser-induced fluorescence detection

Capillary zone electrophoresis (CZE) with laser-induced fluorescence (LIF) detection is shown to constitute a unique technique for the investigation of the interaction between proteases, protease inhibitors and substrates. Under optimized analysis conditions, the formation of a complex between FITC-labelled proteases such as trypsin, plasmin, alpha-chymotrypsin and the (unlabelled) protease inhibitor alpha 2-macroglobulin was studied. This is not possible with UV detection, since under such conditions the complex cannot be distinguished from the unreacted protease inhibitor. Low ratios of FITC bonded to the proteases further complex formation, while high ratios often prevent the reaction. Complex formation shows a strong dependence on the incubation conditions (pH, salt concentration, temperature, incubation time). Once formed, however, the complexes are stable under CZE conditions (e.g., a pH of the electrophoresis buffer of 10.5) for at least 30 min. Treatment with sodium dodecyl sulfate (5 min at 90 degrees C or 30 min at 75 degrees C) does not destroy the complexes, whereas treatment with mercaptoethanol (reduction of disulfide bonds) eliminates the peak from the electropherogram. Both findings argue for the formation of a covalent bond between the protease and the inhibitor during complex formation. Since the reaction of the proteases with alpha 2-macroglobulin does not involve the binding site of the former, a residual proteolytic activity is still observed in the ensuing complex. The extent of the inhibition of the remaining trypsin activity in a trypsin--alpha 2-macroglobulin complex was established to depend on the molecular mass of the second trypsin inhibitor.