Use of ceramic monoliths as stationary phase in affinity chromatography

The use of coated ceramic monoliths as support for affinity chromatography is described. Ceramic monoliths are robust active matrix supports and present a very small pressure drop. Monoliths are coated with a very thin agarose gel layer and activated using a standard activation process for agarose beads. Experiments demonstrate that enzyme adsorption occurs exclusively on the outside surface of the agarose coating since enzyme molecules are too large to fit into the porous matrix. Adsorption and desorption rates are large and production of enzyme per unit monolith volume justifies further exploring this separation process for large throughput operation.     

Préparation et forme sérique de la protéine réactive C de lapin

The preparation of rabbit C-reactive protein (CRP) involves a single step affinity chromatography. This preparation takes advantage of the calcium-dependent affinity of CRP for an agarose gel bearing 2-aminoethanol dihydrogen-phosphate as a ligand. A prior chromatography on agarose gel without the ligand allows the uptake of the serum amyloid P-component (SAP).The CRP prepared according to this method is able to form precipitating complexes in agarose with rabbit lipoproteins. The specificity of these interactions is studied. It is demonstrated that CRP-High Density Lipoproteins (HDL) association produces a second precipitation arc when the pure CRP is revealed by a specific antiserum in agarose. Moreover, CRP in the serum is shown to be in the bound form only, and the binding involves Low Density Lipoproteins (LDL) exclusively.     

Preparation and seric form of rabbit C reactive protein]

The preparation of rabbit C-reactive protein (CRP) involves a single step affinity chromatography. This preparation takes advantage of the calcium-dependent affinity of CRP for an agarose gel bearing 2-aminoethanol dihydrogen-phosphate as a ligand. A prior chromatography on agarose gel without the ligand allows the uptake of the serum amyloid P-component (SAP). The CRP prepared according to this method is able to form precipitating complexes in agarose with rabbit lipoproteins. The specificity of these interactions is studied. It is demonstrated that CRP-High Density Lipoproteins (HLD) association produces a second precipitation arc when the pure CRP is revealed by a specific antiserum in agarose. Moreover, CRP in the serum is shown to be in the bound form only, and the binding involves Low Density Lipoproteins (LDL) exclusively.     

Purification of glycogen debranching enzyme from rabbit muscle using omega-aminoalkyl agarose chromatography

Rabbit muscle glycogen debranching enzyme binds to all of a homologous series of ω-aminoalkyl agaroses. The debrancher can be eluted from ω-aminoethyl and ω-aminobutyl agarose with 0.5 m NaCl, and it desorbs more readily and elutes sooner from ω-aminoethyl agarose than from ω-aminobutyl agarose. No activity is eluted from ω-aminohexyl, octyl, or decyl agaroses. An improved purification procedure has been developed which includes chromatography on ω-aminoethyl agarose. This procedure enables the isolation of over 90% yield of the debranching enzyme from muscle within 3 days.     

Agarose: a possible universal gel exclusion agent

Granulated agarose gels suitable for gel exclusion chromatography of proteins of any molecular weight may now be prepared. This was made possible by the observation that agarose solutions of 16% polysaccharide may be prepared by displacing 8% agarose from solution with 8% polyethylene glycol Mr 6000. The displaced polysaccharide concentrates in a viscous mass occupying half the volume of the original carbohydrate solution. By diluting the displaced polysaccharide with hot watery solutions of electrolyte and allowing the solutions to congeal, gels of any desired concentration, ranging from low to the maximum of 16%, may be prepared.     

Efficient large-scale purification of restriction fragments by solute-displacement ion-exchange HPLC.

Extreme overloading of HPLC columns with sample can create a condition of binding site saturation causing competition and displacement among solutes during column elution. This has been termed solute-displacement chromatography (SD-HPLC). We present an example of this phenomenon for the preparative fractionation and purification of restriction fragments of almost identical size (1337 and 1388 bp) which cannot be resolved by agarose gel electrophoresis. Standard analytical ion-exchange HPLC chromatography failed to separate these fragments from each other and from an unexpectedly early eluting pUC-derived vector fragment of 2.7 kbp. We demonstrate that by intentional overloading of the small (4.6 x 35 mm) non-porous TSK-DEAE HPLC column, hundreds of micrograms of DNA restriction fragments could be resolved and purified in a single HPLC run of less than 30 minutes.     

High-performance molecular sieve chromatography of proteins on agarose columns: the relation between concentration and porosity of the gel

Curves showing the relation between log (molecular weight) and distribution coefficient are presented for proteins subjected to molecular sieve chromatography on crosslinked and non-crosslinked agarose gels of different concentrations. These curves, which facilitate selection of the gel concentration that gives optimal resolution in any particular separation problem, show that the exclusion limit of 5, 9, 12, and 20% agarose gels correspond to protein with molecular weights above 1,000,000, 600,000, 450,000, and 280,000, respectively. Plate numbers have been determined for columns of 20% agarose at different flow rates and bead sizes. Separations of model proteins by high-performance molecular sieve chromatography on agarose beads are shown.     

Selective depletion of small basic non-glycosylated proteins in diabetes.

Degradative rates of small basic non-glycosylated proteins are preferentially enhanced in rat liver cytosol during severe streptozotocin-induced diabetes. Synthetic rates of these classes of proteins are not selectively enhanced in diabetes, so small basic non-glycosylated proteins should be depleted from liver cytosol as a consequence of this disease. To test this hypothesis, proteins were analysed from normal animals, from diabetic animals receiving insulin and from diabetic animals after insulin withdrawal for 3 days. The proteins were separated according to subunit molecular weight by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, according to isoelectric point by isoelectric focusing and according to carbohydrate content by affinity chromatography with concanavalin A linked to agarose. Severe uncontrolled diabetes is associated with the predicted depletion of small basic non-glycosylated proteins from liver cytosol. The preferential degradation and loss of these protein classes may be of considerable physiological importance to the animal.     

中国鲎凝集素的分离纯化及性质研究

 经N-乙酰氨基葡萄糖交联琼脂糖亲和层析及以交联琼脂糖介质的高效液相分子筛层析,从中国鲎细胞溶解物中分离纯化了一种凝集素,其活性比原料鲎试剂提高128倍。鲎凝集素SDS电泳时表现出分子量为69000,和72000的二个亚基。N-乙酰氨基葡萄糖、D-半乳糖,D-甘露糖及岩藻糖等对鲎凝集素凝集鸡红细胞的活性有显著抑制作用,加热60℃,10分钟可使凝集素活性基本丧失。CaCl_2为凝集素活性所必需。鲎凝集素与肺炎球菌C多糖有沉淀反应。     

A large-scale purification of beta-glucuronidase from human liver by immunoaffinity chromatography.

We intend to purify beta-glucuronidase from human liver in a large quantity in order to facilitate the study of its biochemical structure and pathophysiologic roles in cholelithiasis and carcinogenesis. The initial purification procedure involved: (1) liver homogenization, (2) 25-45% saturated ammonium sulfate fractionation, (3) heat denaturation of protein at 56 degrees C, (4) gel filtration with Bio-Gel P-300 gel, (5) anion exchange chromatography with DEAE agarose, (6) cation exchange chromatography with CM agarose, and (7) hydroxyapatite chromatography (overall yield, 1%; overall purification, 169X). The final product was used to immunize rabbits and BALB/c mice for production of polyclonal and monoclonal antibodies, respectively. The antibodies, mainly IgG, were purified by using gamma-Protein A agarose column chromatography. The purified IgG, after periodate oxidation, was coupled to hydrazide gel by formation of a stable covalent hydrazone bond linkage. The new purification procedure involved the initial first three steps, followed by (4) polyclonal IgG immunoaffinity chromatography and (5) monoclonal IgG immunoaffinity chromatography (overall yield, 6.1%; overall purification, 3720X). Polyacrylamide gel electrophoresis indicated minor contaminants in the final product which could be further purified by electroelution. It is concluded that beta-glucuronidase constitutes 0.016 mg per gram of wet liver tissue and can be obtained on a large scale in a highly purified form within a 2-day cycle.     

Drying techniques for the visualisation of agarose‐based chromatography media by scanning electron microscopy

The drying of chromatography resins prior to scanning electron microscopy is critical to image resolution and hence understanding of the bead structure at sub‐micron level. Achieving suitable drying conditions is especially important with agarose‐based chromatography resins, as over‐drying may cause artefact formation, bead damage and alterations to ultrastructural properties; and under‐drying does not provide sufficient resolution for visualization under SEM. This paper compares and contrasts the effects of two drying techniques, critical point drying and freeze drying, on the morphology of two agarose based resins (MabSelect?/d _w ≈85 µm and Capto? Adhere/d _w ≈75 µm) and provides a complete method for both. The results show that critical point drying provides better drying and subsequently clearer ultrastructural visualization of both resins under SEM. Under this protocol both the polymer fibers (thickness ≈20 nm) and the pore sizes (diameter ≈100 nm) are clearly visible. Freeze drying is shown to cause bead damage to both resins, but to different extents. MabSelect resin encounters extensive bead fragmentation, whilst Capto Adhere resin undergoes partial bead disintegration, corresponding with the greater extent of agarose crosslinking and strength of this resin. While freeze drying appears to be the less favorable option for ultrastructural visualization of chromatography resin, it should be noted that the extent of fracturing caused by the freeze drying process may provide some insight into the mechanical properties of agarose‐based chromatography media.     

Molecular morphology of cyanobacterial phycobilisomes

Phycobilisomes were isolated from several cyanobacteria following cell lysis with Triton X-100. They were purified by phosphate precipitation and hydrophobic-interaction chromatography. Their phycobiliprotein compositions were quantitatively determined by application of sets of simultaneous absorbance equations to gel chromatographic separations of the chromoproteins. Phycobilisomes purified from several cyanobacteria had characteristic elution times on agarose gel chromatography. Combining electron microscope observations of phycobilisome structure, phycobiliprotein composition, and agarose gel chromatography estimates of molecular weight permitted the calculation of many details of phycobilisome molecular structure. Complementary chromatic adaptation resulted in a change of phycobilisome composition and structure. The polypeptide compositions of phycobilisomes were examined by sodium dodecyl sulfate-agarose gel chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The phycobilisomes were composed of phycobilipeptides derived from the constituent phycobiliproteins. Higher molecular-weight phycobilipeptide aggregates were also observed. The dominant forces responsible for the maintenance of phycobilisome structure are concluded to be hydrophobic interactions.     

Preparation of electroendosmosis-free agarose gel and exemplification of its use in crossed immunoelectrophoresis

Preparation of electroendosmosis-free agarose is described. The method is based upon the covalent bonding to agarose of a small number of positively charged groups which counterbalance the electroendosmosis-producing effect of the negatively charged groups present in commercial agarose. It is shown how such electroendosmosis-free agarose gel can be combined with advantage with polyacrylamide gels in crossed immunoelectrophoresis.     

Improved protein A resin for antibody capture in a continuous countercurrent tangential chromatography system

Continuous countercurrent tangential chromatography (CCTC) enables steady-state continuous bioprocessing with low-pressure operation and high productivity. CCTC has been applied to initial capture of monoclonal antibodies (mAb) from clarified cell culture harvest and postcapture polishing of mAb; however, these studies were performed with commercial chromatography resins designed for conventional column chromatography. In this study, a small particle size prototype agarose resin (20–25 µm) with lower cross-linking was co-developed with industrial partner Purolite and tested with CCTC. Due to increased binding capacity and faster kinetics, the resulting CCTC process showed more than a 2X increase in productivity, and a 2X reduction in buffer consumption over commercial protein A resins used in previous CCTC studies, as well as more than a 10X productivity increase versus conventional column operation. Single-pass tangential flow filtration was integrated with the CCTC system, enabling simple control of eluate concentration. A scale-up exercise was conducted to provide a quantitative comparison of CCTC and batch column chromatography. These results clearly demonstrate opportunities for using otherwise unpackable soft small particle size resins with CCTC as the core of a continuous bioprocessing platform.     

Immunoaffinity isolation of apolipoprotein E-containing lipoproteins

Discrete apolipoprotein E-containing lipoproteins can be identified when EDTA plasma is fractionated on columns of 4% agarose. The present study has demonstrated, by physical and metabolic criteria, that these apolipoprotein E-containing lipoprotein subclasses may be further isolated by immunoaffinity chromatography. Whole plasma was first bound to an anti-apolipoprotein E immunoadsorbent prior to gel filtration on 4% agarose. After elution from the affinity column and dialysis, the bound fraction was chromatographed on 4% agarose. Discrete subfractions of apolipoprotein E could be demonstrated within elution volumes similar to those observed in the original plasma. When whole plasma was first submitted to gel filtration and the apolipoprotein E-containing lipoproteins of either intermediate- or of high-density lipoprotein (HDL) size were subsequently bound to anti-apolipoprotein E columns, the bound eluted fractions maintained their size and physical properties as shown by electron microscopy and by rechromatography on columns of 4% agarose. The metabolic integrity of apolipoprotein E-containing very-low-density lipoproteins (VLDL) was examined by coinjection into a cynomolgus monkey of 125I-labeled apolipoprotein E-rich and 131I-labeled apolipoprotein E-deficient human VLDL which had been separated by immunoaffinity chromatography. The plasma specific activity time curves of the apolipoprotein B in VLDL, intermediate-density (IDL) and low-density (LDL) lipoproteins demonstrated rates of decay and precursor-product relationships similar to those obtained after injection of whole labeled VLDL, supporting the metabolic integrity of VLDL isolated by immunoaffinity chromatography.     

A rapid method for the purification of RNA polymerase holoenzyme from Escherichia coli

A method is described for the rapid purification of RNA polymerase holoenzyme from small amounts of Escherichia coli cells. Chromatography of a crude extract on a single-stranded DNA agarose column followed by gel filtration chromatography gave 95% pure holoenzyme. The enzyme had kinetic characteristics on T7 DNA identical to those of RNA polymerase purified by other more laborious procedures.     

Agar and agarose biotechnological applications

Agar, a phycocolloid obtained commercially from species of Gelidium and Gracilaria, has been known for several centuries; its earliest industrial application was in the preparation of solid microbiological media. The numerous techniques available for the purification of agar affect the characteristics of bacterial-grade agar. The availability of agarose, that fraction of agar with the lowest possible charge, has enhanced the utilzation of this phycocolloid. The process of gelation of agarose is discussed and the applications of agarose gels in different types of chromatography are summarized. Agarose has many and diverse important applications in biotechnology. These uses, and newly-developed ones, can be expected to increase the demands for high-quality agarose in the rapidly expanding field of biotechnology.     

"Clickable" agarose for affinity chromatography

Successful purification of biological molecules by affinity chromatography requires the attachment of desired ligands to biocompatible chromatographic supports. The Cu(I)-catalyzed cycloaddition of azides and alkynes-the premier example of "click chemistry"-is an efficient way to make covalent connections among diverse molecules and materials. Both azide and alkyne units are highly selective in their reactivity, being inert to most chemical functionalities and stable to wide ranges of solvent, temperature, and pH. We show that agarose beads bearing alkyne and azide groups can be easily made and are practical precursors to functionalized agarose materials for affinity chromatography.     

A simple, one-step chromatographic procedure for the purification of lysozyme

A rapid method for the purification of lysozyme (mucopeptide N-acetyl-muramoylhydrolase, EC 3.2.1.17) from hen egg-white has been devised. It was that gel filtration chromatography on agarose columns can be used selectively to purify lysozyme, due to the fact that this protein interacts with the agarose matrix and elutes later than the corresponding total volume for the column. Thus, lysozyme is directly obtained in a relatively pure form and with a high specific activity. In principle, this simple method can be used to prepare lysozymes from other sources.     

Selective protein transport: Identity of the solubilized phosvitin receptor from chicken oocytes

By two independent methods, the solubilized receptor for phosvitin (PV) has a subunit MW of 116K. Affinity chromatography, showed that only 2 of the more than 25 proteins present in the total detergent solubilized oocyte membrane extract were retained on a PV–agarose column. These proteins of MW of 116K and 100K could be eluted from PV–agarose with free PV. By gel exclusion chromatography, the receptor-¹²⁵I-PV complexes elute in the void volume of a Biogel A-1.5 column. When these void fractions were assayed by SDS-PAGE only a single protein of MW of 116K was observed in addition to ¹²⁵I-PV.