Affinity purification of aprotinin from bovine lung

An affinity protocol for the purification of aprotinin from bovine lung was developed. To simulate the structure of sucrose octasulfate, a natural specific probe for aprotinin, the affinity ligand was composed of an acidic head and a hydrophobic stick, and was then linked with Sepharose. The sorbent was then subjected to adsorption analysis with pure aprotinin. The purification process consisted of one step of affinity chromatography and another step of ultrafiltration. Then purified aprotinin was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, trypsin inhibitor activity, gel‐filtration, and thin‐layer chromatography analysis. As calculated, the theoretical maximum adsorption (Q_max) of the affinity sorbent was 25 476.0 ± 184.8 kallikrein inactivator unit/g wet gel; the dissociation constant of the complex “immobilized ligand‐aprotinin” (K_d) was 4.6 ± 0.1 kallikrein inactivator unit/mL. After the affinity separation of bovine lung aprotinin, reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and gel‐filtration chromatography revealed that the protein was a single polypeptide, and the purities were ～ 97 and 100%, respectively; the purified peptide was also confirmed with aprotinin standard by gel‐filtration chromatography and thin‐layer chromatography. After the whole purification process, protein, and bioactivity recoveries were 2.2 and 92.6%, respectively; and the specific activity was up to 15 907.1 ± 10.2 kallikrein inactivator unit/mg.     

Affinity chromatographic purification of lentil lectin using immobilized yeast cells

A model system for evaluating macroporous supports containing immobilized whole cells in affinity Chromatographic applications is described. Whole cells were immobilized in a polyacrylamide network in a two-step polymerization process. The affinity system discussed consists of immobilized cells ofSaccharomyces cervisiae in the purification of lentil lectin fromLens culinaris.     

Affinity purification of proteins using expanded beds.

The use of expanded beds of affinity adsorbents for the purification of proteins from feedstocks containing whole or broken cells is described. It is demonstrated that such feedstocks can be applied to the bed without prior removal of particulate material by centrifugation or filtration thus showing considerable potential for this approach in simplifying downstream processing flow-sheets. A stable, expanded bed can be obtained using simple equipment adapted from that used for conventional packed bed adsorption and chromatography processes. Circulation and mixing of the adsorbent particles is minimal and liquid flow through the expanded bed shows characteristics similar to those of plug flow. Frontal analysis performed with the highly selective affinity system involving the adsorption of human polyclonal immunoglobulin G onto Protein A Sepharose Fast Flow indicate that the adsorption performance of the expanded bed is similar to that achieved when the same amount of adsorbent is used in a packed configuration at the same volumetric flow-rate. The adsorption performance of the expanded bed was not diminished when adsorption was carried out in the presence of intact yeast cells. Batch adsorption experiments also indicated that the adsorption characteristics of the affinity system were not greatly altered in the presence of cells in contrast to results from a less selective ion-exchange system. An expanded bed of Cibacron Blue Sepharose Fast Flow was used to purify phosphofructokinase from feedstock of disrupted yeast prepared by high pressure homogenisation without the need for prior removal of particulate material. The potential for the use of expanded beds in large scale purification systems is discussed.     

Affinity purification and affinity characterization of carbohydrate-binding proteins in bovine kidney.

Ca(2+)-dependent carbohydrate-binding proteins were purified from bovine kidney by two-step affinity chromatography on fetuin and heparin columns and subsequent anion-exchange high-performance liquid chromatography. On sodium dodecyl sulphate-polyacrylamide gel electrophoresis, the purified fraction gave two protein bands corresponding to proteins of relative molecular mass 33,000 (p33) and 41,000 (p41), respectively. Although the proteins had no haemogglutinating activities towards human and rabbit erythrocytes, their carbohydrate-binding activity was examined by a newly developed method using horseradish peroxidase (HRP) and/or biotin-labelled glycoconjugates as affinity probes. They could bind in a Ca(2+)-dependent manner to labelled fetuin and heparin in a specific and dose-dependent manner by solid-phase assay after immobilization on plastic plate surface. Inhibition assay of the binding revealed that N-acetylneuraminic acid is the most potent inhibitor of the proteins among the monosaccharides tested. Fucoidin and heparan sulphate most strongly inhibited the binding of the proteins to labelled heparin. Direct binding assay to acidic glycolipids prepared from bovine kidney showed that the proteins react with the ganglioside fraction but not with sulphatide [Gal(3-SO4) beta 1-1Cer]. These results indicated that the purified proteins have a significant affinity to charged oligosaccharides linking to glycoproteins, glycolipids and charged polysaccharides in a Ca(2+)-dependent manner.     

Affinity chromatographic purification of immunoglobulin M antibodies utilizing immobilized mannan binding protein.

A method is described for the rapid and efficient affinity chromatographic purification of murine monoclonal immunoglobulin M (IgM) which utilizes immobilized rabbit mannan binding protein (MBP). This solid-phase matrix is shown to bind IgM-class antibodies from a variety of species. Conditions reported show a binding capacity of IgM from murine ascites of nearly 1 mg/ml of immobilized MBP support. The prepared gel is shown to possess an ability to bind not only mouse IgM, but also human and bovine IgM, although with a lesser affinity. The matrix can be regenerated and reused at least ten times without any apparent loss of binding capacity or specificity. Mouse monoclonal IgM purified from ascites fluid using this method is greater than 95% pure as shown by high-performance liquid chromatography analysis.     

Affinity purification of antibodies using immobilized FB domain of protein A.

A continuous method for the efficient digestion of protein A into active fragments (FB, Mr = 7000) using immobilized trypsin was developed. These fragments originate from almost identical five-repeated monovalent Fc-binding units of 58 residues each. The fragments obtained were found to be similar to the recently described genetically engineered fragment B. Antibody-binding characteristics of the FB domain and also of intact protein A, immobilized on to adipic dihydrazide-modified Eupergit CB6200 beads, were investigated. Based on the experimental data obtained, a high-performance liquid chromatographic column containing C30N Eupergit C-immobilized FB domain was prepared and its performance in antibody purification was compared with that of Eupergit C-immobilized intact protein A.     

Ion-exchange high-performance liquid chromatographic purification of bovine cardiac and rabbit skeletal muscle troponin subunits

Bovine cardiac and rabbit skeletal troponin complexes were separated into their respective subunits employing high-performance liquid chromatographic (HPLC) techniques on CM-300 and Q-300 ion-exchangers. Bovine cardiac and rabbit skeletal subunits were separated on the strong anion-exchanger, Q-300, in 8 M urea, 50 mM Tris, 2 mM EGTA, 0.5 mM dithiothreitol, pH 7.5, employing a linear salt gradient and on the weak cation-exchanger, CM-300, in 8 M urea, 50 mM potassium dihydrogen phosphate, 2 mM EGTA, 0.5 mM dithiothreitol, pH 6.5, using a linear salt gradient. To obtain complete purification of all components of troponin both ion-exchangers were required. The initial separation of troponin was carried out on the strong anion-exchanger followed by weak cation-exchange chromatography of the troponin I collected from the strong anion-exchange column. The troponin T subunits obtained from Q-300 chromatography demonstrated heterogeneity (three components: T1, T2 and T3) while the troponin I collected from both sources on the Q-300 column were both resolved into major doublets (I1 and I2) when rechromatographed on the CM-300 column. The three troponin T fractions and two troponin I fractions isolated from ion-exchange HPLC were examined by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis to confirm that the heterogeneity was due to differences in charge and not molecular weight. These results were in agreement with the charge differences observed from retention times on ion-exchange HPLC. When comparing the same troponin subunit from different muscle sources, considerable differences in the content of charged amino acid residues were also observed.     

Liquid chromatographic purification and detection of anabolic compounds.

The role of liquid chromatography within methods of analysis for steroids, related compounds and beta-agonists in biological samples is discussed. Special attention is given to the application of liquid chromatography in sample preparation and extract clean-up. Different forms of liquid chromatography, including immunoaffinity chromatography, are compared and evaluated. Methods for confirmation based on gas chromatography-mass spectrometry and cryotrapping Fourier transform infrared spectrometry are discussed.     

Affinity purification of metalloprotease from marine bacterium using immobilized metal affinity chromatography

In this study, an efficient affinity purification protocol for an alkaline metalloprotease from marine bacterium was developed using immobilized metal affinity chromatography. After screening and optimization of the affinity ligands and spacer arm lengths, Cu‐iminmodiacetic acid was chosen as the optimal affinity ligand, which was coupled to Sepharose 6B via a 14‐atom spacer arm. The absorption analysis of this medium revealed a desorption constant K_d of 21.5 μg/mL and a theoretical maximum absorption Q_max of 24.9 mg/g. Thanks to this affinity medium, the enzyme could be purified by only one affinity purification step with a purity of approximately 95% pure when analyzed by high‐performance liquid chromatography and reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis. The recovery of the protease activity reached 74.6%, which is much higher than the value obtained by traditional protocols (8.9%). These results contribute to the industrial purifications and contribute a significant reference for the purification of other metalloproteases.     

Affinity chromatography in purification of A1 adenosine receptors.

Purification of A1 adenosine receptor of rat brain membranes was performed using a newly developed affinity gel employing xanthine amine congener (XAC) as an immobilized ligand. The A1 adenosine receptor was solubilized with digitonin-cholate from brain membranes and then purified by a sequential use of affinity chromatography on XAC-agarose, hydroxyapatite chromatography and reaffinity chromatography on XAC-agarose. The A1 adenosine receptor was purified ca. 45,000-fold with a yield of 5%. The final receptor preparation gave a single broad band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a Mr approximately 34,000. This band was also shown to be specifically labelled with an affinity labelling reagent for A1 adenosine receptors. This purification method was also applicable for the complete purification of A1 adenosine receptors from rat testis and human brain membranes.     

Affinity purification of viral protein having heterogeneous quaternary structure: Modeling the impact of soluble aggregates on chromatographic performance

Prokaryote-expressed polyomavirus structural protein VP1 with an N-terminal glutathione-S-transferase tag (GST-VP1) self-assembles into pentamer structures that further organize into soluble aggregates of variable size (3.4 × 10²–1.8 × 10⁴ kDa) [D.I. Lipin, L.H.L. Lua, A.P.J. Middelberg, J. Chromatogr. A 1190 (2008) 204]. The adsorption mechanism for the full range of GST-VP1 soluble aggregates was described assuming a dual-component model [T.Y. Gu, G.J. Tsai, G.T. Tsao, AICHE J. 37 (1991) 1333], with components differentiated by size, and hence pore accessibility, rather than by protein identity. GST-VP1 protein was separated into two component groups: aggregates small enough to access resin pores (LMW: 3.4 × 10²–1.4 × 10³ kDa) and aggregates excluded from the resin pores (HMW: 9.0 × 10²–1.8 × 10⁴ kDa). LMW aggregates bound to resin at a higher saturation concentration (29.7 g L⁻¹) than HMW aggregates (13.3 g L⁻¹), while the rate of adsorption of HMW aggregates was an order of magnitude higher than for LMW aggregates. The model was used to predict both batch and packed bed adsorption of GST-VP1 protein in solutions with known concentrations of HMW and LMW aggregates to Glutathione Sepharose HP resin. Asymmetrical flow field flow fractionation with UV absorbance was utilized in conjunction with adsorption experimentation to show that binding of HMW aggregates to the resin was strong enough to withstand model-predicted displacement by LMW aggregates. High pore concentrations of LMW aggregates were also found to significantly inhibit the diffusion rate of further protein in the resin pores. Additional downstream processing experimentation showed that enzymatic cleavage of LMW aggregates to remove GST tags yields more un-aggregated VP1 pentamers than enzymatic cleavage of HMW aggregates. This model can be used to enhance the chromatographic capture of GST-VP1, and suggests an approach for modeling chromatographic purification of proteins that have a range of quaternary structures, including soluble aggregates.     

Production of milligram quantities of affinity tagged-proteins using automated multistep chromatographic purification

A new chromatography system, AKTAxpress (GE Healthcare, Amersham Biosciences, Uppsala, Sweden) has been designed to meet the demand for high-throughput purification of proteins in structural genomics and drug discovery. The system offers a number of automated multistep purification protocols for affinity-tagged proteins. All protocols start with affinity chromatography followed by combinations of desalting, ion exchange chromatography and gel filtration. As an option, tag removal can be included in the purification protocols. Up to 16 proteins can be purified per day and the yield can be as high as 50 mg of each protein at > 90% purity. To highlight the versatility of the system, this paper presents several case studies; purifications of hexahistidine- and glutathione S-transferase-tagged proteins using different protocols, automated on-column tag cleavage and optimization studies for a hexahistidine-tagged kinase.     

Selective high-performance liquid chromatographic purification of bispecific monoclonal antibodies.

The recent development of improved production techniques for bispecific monoclonal antibodies (biMAbs) has significantly increased interest in specific purification procedures. In this investigation, a general high-performance liquid chromatographic (HPLC) purification method is proposed that allows highly purified biMAbs to be obtained from mouse ascites fluid containing a mixture of different antibodies, i.e., parental MAbs, active biMAb and a mixture of randomly assembled heavy and light chains. Proteins from ascites fluid were precipitated with ammonium sulphate and applied to a high-performance protein A column to separate the total immunoglobulin fraction. BiMAbs were isolated from other immunoglobulins by two subsequent passages through a high-performance hydroxyapatite (HPHT) column. This purification protocol combines specificity of protein A for immunoglobulin G (IgG) and high selectivity of hydroxyapatite for different IgG idiotypes. All purification steps were performed rapidly and reliably by HPLC. This method was applied to the purification of six different biMAbs with consistently high yields, purity and homogeneity. This general purification method may prove extremely valuable when highly pure preparation of biMAbs is required, as for in vivo use.     

Affinity chromatography for the purification of therapeutic proteins from transgenic maize using immobilized histamine

Plant molecular pharming is a technology that uses plants as bioreactors to produce recombinant molecules of medical and veterinary importance. In the present study, we evaluated the ability of histamine (HIM), tryptamine (TRM), phenylamine (PHEM) and tyramine (TYRM) coupled to Sepharose CL-4B via a 1,4-butanediol diglycidyl ether spacer to bind and purify human monoclonal anti-HIV antibody 2F5 (mAb 2F5) from spiked maize seed and tobacco leaf extracts. Detailed studies were carried out to determine the factors that affect the chromatographic behaviour of mAb 2F5 and also maize seed and tobacco leaf proteins. All affinity adsorbents showed a reduced capacity to bind and a reduced ability to purify proteins from tobacco extract compared to maize extract. Under optimal conditions, HIM exhibited high selectivity for mAb 2F5 and allowed a high degree of purification (>95% purity) and recovery (>90%) in a single step with salt elution (0.4 M KCl) from spiked maize seed extract. Analysis of the purified antibody fraction by ELISA and Western blot showed that the antibody was fully active and free of degraded variants or modified forms. The efficacy of the system was assessed further using a second therapeutic antibody (human monoclonal anti-HIV antibody mAb 2G12) and a therapeutic enzyme (alpha-chymotrypsin). HIM may find application in the purification of a wide range of biopharmaceuticals from transgenic plants.     

Affinity column purification of amylases on protein inhibitors from wheat kernel.

An affinity column was devised for the purification of a large number of amylases inhibited by the albumin from wheat kernel. The procedure involved linking the protein inhibitors from wheat to Sepharose and then specifically eluting the amylase adsorbed to the gel with a high concentration of maltose. By this procedure, the amylases from Tenebrio molitor L. (yellow mealworm) larvae and chicken pancreas were purified to homogeneity with good yields for the first time, as shown by both alkaline and acidic electrophoresis. Human saliva alpha-amylase, purified by the same procedure, showed specific activity and electrophoretic patterns similar to those obtained by other workers with different techniques.     

Affinity resins for the purification of opioid-binding materials

Two new affinity resins for the purification of opioid-binding materials were prepared. One was AH-Sepharose coupled with [D-Ala2, D-Leu5]enkephalin and the other was AF-Amino Toyopearl with [D-Ala2, Leu5]enkephalin. Solubilized-opioid receptors from rat brain were treated with these affinity resins and the materials with opioid-binding activities were purified. On sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, the purified materials showed one major band with a molecular weight of 62000-64000. The results suggested that the prepared resins are useful tools for the purification of opioid receptors.     

Affinity purification of proteinases by a combination of immobilized peptidyl aldehyde and semicarbazone.

D-Phe-Phe-argininal semicarbazone and Tyr-Gly-Gly-Phe-Leu-Arg-argininal semicarbazone were prepared using the solution phase synthesis method and characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. The tripeptide and heptapeptide semicarbazones were individually immobilized on affi-Gel 15 resulting in two affinity columns called S3 and S7, respectively. A third affinity column was obtained by hydrolysing the semicarbazone moiety in column S3 to aldehyde (column A3). Serine proteinases such as trypsin or rat plasma kallikrein almost quantitatively bind to either S3 or A3 affinity columns. Under optimized conditions, more than 97% of trypsin bound to both columns S3 and A3. At a lower ionic strength and higher pH, 80-85% of rat plasma kallikrein bound to the same columns. Elution of both enzymes was achieved using mild conditions at near neutral pH and in the presence of a small amount of denaturant. Both proteinases were identified and characterized by high-performance liquid chromatography, sodium dodecylsulphate polyacrylamide gel electrophoresis and by their substrate specificity and inhibition profiles. A single purification (six-to seven-fold) step using either column S3 or A3 allowed the preparation of pure trypsin from commercial sources. Starting from rat plasma partially purified by a phenyl boronate column, fractionation on the S3 column allowed approximately an 87-fold purification of rat plasma kallikrein. However, serial purification of rat plasma kallikrein on column S7 followed by column A3 resulted in a purification factor of about 455.     

Affinity purification of plasminogen by radial-flow affinity chromatography

A method for the purification of plasminogen using immobilized L-lysine on a membrane, the whole system being constructed in a radial flow cartridge, is described. Human plasma was applied to the cartridge at 20 ml/min. The results showed that under the chromatographic conditions chosen, in a single pass, greater than 85% recovery of plasminogen was attained with a 110-fold increase in specific activity.     

Enzymatic synthesis and chromatographic purification of lignan glucuronides

Enterolactone, enterodiol and secoisolariciresinol were conjugated with glucuronic acid by solubilized rabbit liver microsomal UDP-glucuronyltrasnferase. The monoglucuronide conjugate of all three substrates was formed and its identity confirmed by nuclear magnetic resonance (NMR) spectroscopy. Analytical high pressure liquid chromatography (HPLC) and NMR spectroscopy indicated conjugation with glucuronic acid to occur at several positions in the molecule. The enzymatic conjugation was monitored by analytical capillary isotachophoresis (ITP). The Km-values for enterlactone, enterodiol, and secoisolariciresinol were calculated to be 0.30, 0.23, and 0.22 mmol/l respectively.