Affinity Chromatography Resin Research Articles

Synthesis of γ-Phosphate-Linked Nucleoside Affinity Chromatography Resins for Protein Purification, Including Ribonucleoside Triphosphate Reductase

Seven nucleotides linked through the γ-phosphate to diamine hydrocarbons were synthesized and coupled to Sepharose for use in protein purification affinity chromatography. The synthesis involved converting the nucleotides to nucleoside-5′-trimetaphosphates using dicyclohexyl carbodiimide, followed by nucleophilic ring opening of the trimetaphosphate with an α,ω-diamino hydrocarbon to generate a γ-phosphoamide linkage in each nucleotide.

On the adsorption of hyaluronan and ICAM-1 to modified hydrophobic resins

Hyaluronan is a negatively charged glycosaminoglycan that occurs in connective tissue and has a wide range of mechanical and cell biological functions. The purpose of this study was to utilize affinity chromatography resins for purification of detergent (Triton X-100) solubilized hyaluronan binding proteins from liver, the major organ of hyaluronan clearance from the blood. However, during these studies we made the unexpected finding that hyaluronan binds to Sepharose substituted with a hexamethylene chain, a commonly used spacer arm in affinity chromatography resins, capped with either a terminal primary amine or a terminal acetoamido group. Hyaluronan did not bind the hydrophobic resins hexyl- or octyl-Sepharose under the same conditions. It was also found that rat liver intercellular adhesion molecule-1 binds to resins containing the hexamethylene spacer arm, an interaction which could be inhibited with free hyaluronan oligosaccharides. Finally, we have determined that resins with ethylene spacer arms show no affinity for hyaluronan and can therefore be used to immobilize hyaluronan for chromatography of hyaluronan binding proteins. By using this resin we have purified two proteins of approximately 200 and 400 kDa from rat liver endothelial cells. In summary, this study demonstrates the efficacy of certain “capped-hydrophobic” resins for binding hyaluronan; these resins may provide a novel means for the study and/or purification of this glycosaminoglycan. This study further demonstrates the importance of the careful design of appropriate affinity columns for the specific purification of hyaluronan binding proteins.

Synthesis, cytotoxic properties and effects on early and late gene induction of a chimeric diphtheria toxin-leukemia-inhibitory factor protein.

Leukemia-inhibitory factor (LIF) is a neuropoietin able to regulate the differentiation and the survival of many cell types, which include some neuronal populations. The present study describes the genetic construction, expression, purification and properties of a diphtheria-toxin-related LIF gene fusion in which the native receptor-binding domain of diphtheria toxin was replaced with a gene encoding human LIF. The fusion protein expressed from the chimeric tox gene was designated DT-(1-389)-LIF-(2-184)-peptide. This fusion protein has a deduced molecular mass of 65980 Da and is formed by fusion of the first 389 amino acids of diphtheria toxin to amino acids 2-184 of mature human LIF, using a linker of 34 amino acids that includes six consecutive histidine residues. The latter span allows for single-step purification of the fusion protein by Ni(2+)-resin affinity chromatography. This linker provides a high degree of flexibility between the diphtheria toxin and LIF domains, thereby permitting aggregation-free refolding of the chimeric protein while bound to the affinity column. Both LIF and DT-(1-389)-LIF-(2-184)-peptide induced the phosphorylation of CLIP1 and CLIP2 in LIF-responsive neuroblastoma SH-N-BE cells. DT-(1-389)-LIF-(2-184)-peptide was selectively cytotoxic for cultured neuroblastoma cells bearing the LIF receptor, and for sympathetic neurons. The cytotoxic action of DT-(1-389)-LIF-(2-184)-peptide, like that of native diphtheria toxin, required receptor-mediated endocytosis, passage through an acidic compartment, and delivery of an ADP-ribosyltransferase to the cytosol of target cells. The latter point was confirmed by the fact that, while both LIF and DT-(1-389)-LIF-(2-184)-peptide increased c-fos mRNA expression in SH-N-BE cells, only LIF induced proenkephalin and c-fos promoter activities in cells transiently transfected with c-fos-chloramphenicol acetyltransferase and proenkephalin-chloramphenicol acetyltransferase fusion genes. Mutational analysis suggested that the C-terminal helix (helix D) of human LIF may, in part, constitute or contribute to the active site for LIF receptor binding and cell activation. The cytotoxic properties of DT-(1-389)-LIF-(2-184)-peptide may be useful in selectively depleting neuronal and immune cell populations that express the LIF beta receptor.

Ancylostoma caninum anticoagulant peptide: cloning by PCR and expression of soluble, active protein in E. coli

Ancylostoma caninum Anticoagulant Peptide (AcAP) is the major anticoagulant activity present in extracts of adult Ancylostoma caninum hookworms. This 8.7 kDa protein is a potent and specific inhibitor of human coagulation factor Xa. Using PCR, we have isolated a cDNA encoding for AcAP from an adult A. caninum cDNA library. The 5′ end of the AcAP cDNA was identified by reverse transcription PCR (RT-PCR) using A. caninum cDNA and a 5′ primer corresponding to a nematode spliced leader sequence. The AcAP cDNA was expressed in E. coli using a prokaryotic expression vector, and the recombinant fusion protein (rAcAP) was purified to homogeneity using nickel resin affinity chromatography and reverse phase HPLC. Purified rAcAP is comparable to the native protein in inhibitory activity, with an apparent equilibrium inhibitory dissociation constant ( K i ∗ ) for the inhibition of factor Xa of 265+ −71 pM . The purified protein also prolongs the prothrombin and partial thromboplastin times of human plasma in a dose dependent manner.

Purification and Characterization of Decorin Core Protein Expressed inEscherichia colias a Maltose-Binding Protein Fusion

Decorin is a small leucine-rich proteoglycan which is a component of the extracellular matrix of many connective tissues. We have developed a method for expression and purification of decorin as a fusion protein inEscherichia coli.A PCR product coding for the 330-amino-acid-residue secreted form of bovine decorin core protein was cloned into the vector pMal-c for expression inE. colias a maltose-binding protein (MBP) fusion. Expressed MBP–decorin tended to form insoluble aggregates resistant to degradation byE. coliintracellular proteases. Fusion protein was therefore solubilized from bacterial inclusion bodies using guanidine HCl and refolded by dilution of the chaotrope to minimally denaturing conditions and disulfide shuffling. Final purification included amylose resin affinity chromatography and size exclusion chromatography. The 79-kDa MBP–decorin fusion protein could be completely cleaved by factor Xa protease in 24 h to yield 43-kDa MBP and 36-kDa decorin core protein. The decorin core protein was separated from MBP and factor Xa protease by DEAE–Sephacel chromatography. Using a solid-phase assay, we have characterized its binding affinity for extracellular matrix components known to interact with tissue-derived decorin including collagen type VI, collagen type I, and fibronectin. The purification and refolding protocol described here may be generally applicable to bacterial expression of other members of this growing family of related small leucine-rich proteoglycan core proteins.

One-step affinity purification of bacterially produced proteins by means of the “Strep tag” and immobilized recombinant core streptavidin

The “Strep tag” is a nine amino acid peptide with intrinsic streptavidin-binding activity. If this sequence is genetically fused to the C-terminus of a polypeptide the recombinant protein can be directly purified by affinity chromatography from the host cell extract on immobilized streptavidin. However, variations were observed in the suitability of different commercial streptavidin-agarose preparations for this purpose. Therefore, the influence of the source of streptavidin, the coupling chemistry, and the nature of the affinity chromatography resin was investigated. A procedure was developed for the production of recombinant core streptavidin in Escherichia coli, followed by its efficient refolding and purification with an overall yield of up to 140 mg functional protein per 1 1 bacterial culture. When coupled to activated CH-Sepharose 4B this truncated form of streptavidin showed a performance in the affinity chromatography of Strep tag fusion proteins that was superior to all other combinations tested. In contrast to its conventional preparation from Streptomyces strains the recombinant core streptavidin was produced without a proteolytic processing step. Thus, deleterious effects during the streptavidin affinity purification of proteins due to residual proteolytic activity in the immobilized streptavidin were avoided. The versatility of the optimized purification system was demonstrated with five different Strep tag fusion proteins.

Purification of two active fusion proteins of the Na +-dependent citrate carrier of Klebsiella pneumoniae

The sodium-ion-dependent citrate carrier of Klebsiella pneumoniae (CitS) was purified by means of bioengineerical methods. By fusing the biotin acceptor domain of the α-subunit of the oxaloacetate decarboxylase of K pneumoniae to the C-terminus of CitS, purification of the carrier was achieved by use of a monomeric avidin-Sepharose column. Additionally, we were able to purify a CitS-protein with an N-terminal histidine-tag by immobilized metal chelate affinity chromatography (with Ni 2+-nitrilotriacetic acid-(NTA-) resin). Both purified fusion proteins showed citrate transport activity after reconstitution into liposomes by the freeze/thaw/sonication procedure.

Isolation and purification of proteoglycans.

Purification of a protein typically involves development of a quantitative assay to track protein integrity (e.g. enzyme activity) during subsequent isolation steps. The generalized procedure involves choosing the source of the protein, defining extraction conditions, developing bulk purification methods followed by refined, more selective methods. The purification of proteoglycans is often complicated by a) limited source quantities, b) necessity of chaotropic solvents for efficient extraction, c) their large molecular size and d) lack of defined functions to enable purity (i.e. activity, conformation) to be assessed. Because the usual goal of proteoglycan purification is physical characterization (intact molecular weight, core protein and glycosaminoglycan class and size), the problems of a suitable assay and/or native conformation are avoided. The 'assay' for tracking proteoglycan isolation typically utilizes uronic acid content or radiolabel incorporation as a marker. Once extracted from their cellular/extracellular environment, proteoglycans can be isolated by density gradient centrifugation and/or column chromatography techniques. Recent advances in the composition of chromatographic supports have enabled the application of ion-exchange, gel permeation, hydrophobic interaction and affinity chromatography resins using efficient high-pressure liquid chromatography to proteoglycan purification.

Chaperonins groEL and groES promote assembly of heterotetramers (alpha 2 beta 2) of mammalian mitochondrial branched-chain alpha-keto acid decarboxylase in Escherichia coli.

We have investigated the possible role of chaperonins groEL and groES in the folding and assembly of heterotetramers (alpha 2 beta 2) of mammalian mitochondrial branched-chain alpha-keto acid decarboxylase (E1) in Escherichia coli. The mature E1 alpha subunit fused to maltose-binding protein (MBP) was coexpressed with mature E1 beta on the same vector in ES- and EL- mutant strains. Only small or trace amounts of active E1 component were obtained. Cotransformation of the ES- mutant host with a second vector overexpressing groEL and groES resulted in a greater than 500-fold increase in E1-specific activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the content of both MBP-E1 alpha and E1 beta polypeptides was markedly increased in the presence of overexpressed chaperonin proteins. The time course studies showed that the increase in E1-specific activity and subunit levels correlated with the increase in groEL and groES until the concentration of the chaperonins reached a saturating level in the cell. The functional MBP-E1 fusion protein from ES- double transformants were purified by amylose resin affinity chromatography. The MBP moiety was removed by subsequent digestion with Factor Xa endoprotease, followed by Sephacryl S-300HR chromatography. It was found that E1 alpha and E1 beta assembled into an active 160-kDa species, which was consistent with the alpha 2 beta 2 structure of E1. The present results demonstrate that chaperonins groEL and groES promote folding and assembly of heterotetrameric proteins of mammalian mitochondrial origin.

Solubilization and Purification of Melatonin Receptors from Lizard Brain*

Melatonin receptors in lizard brain were identified and characterized using 125I-labeled melatonin ([125I]MEL) after solubilization with the detergent digitonin. Saturation studies of solubilized material revealed a high affinity binding site, with an apparent equilibrium dissociation constant of 181 +/- 45 pM. Binding was reversible and inhibited by melatonin and closely related analogs, but not by serotonin or norepinephrine. Treatment of solubilized material with the non-hydrolyzable GTP analog, guanosine 5'-(3-O-thiotriphosphate) (GTP-gamma-S), significantly reduced receptor affinity. Gel filtration chromatography of solubilized melatonin receptors revealed a high affinity, large (Mr 400,000) peak of specific binding. Pretreatment with GTP-gamma-S before solubilization resulted in elution of a lower affinity, smaller (Mr 150,000) peak of specific binding. To purify solubilized receptors, a novel affinity chromatography resin was developed by coupling 6-hydroxymelatonin with Epoxy-activated Sepharose 6B. Using this resin, melatonin receptors were purified approximately 10,000-fold. Purified material retained the pharmacologic specificity of melatonin receptors. These results show that melatonin receptors that bind ligand after detergent treatment can be solubilized and substantially purified by affinity chromatography.

Renin in human fetal lung--a biochemical and immunohistochemical study.

Human fetal lung homogenates contain an inactive form of renin which may be revealed by trypsin treatment. When activated, this form of renin has some biochemical similarities with fetal kidney renin: the pH optimum of fetal lung renin is approximately 6.5; it is bound by Affigel Blue affinity chromatography resin; and is inhibited by a monoclonal antibody (R-3-36-16) raised to human kidney renin. Inactive renin, partially-purified from both fetal kidney and lung, differs from this in that the renal form is of low-molecular weight (LMW, 45,000 daltons), whereas that from fetal lung is of high molecular weight (HMW, 58,000 daltons). Using a sensitive alkaline phosphatase-anti-alkaline phosphatase (APAAP) procedure with a polyclonal anti-renin antibody (R-15), immunoreactive renin in fetal lung was found in vessels in mesenchyme between airways. The pattern of staining was distributed similarly to Factor VIII-related antigen, suggesting localization in endothelial cells.

Development of an affinity chromatography resin for the purification of carcinogen binding proteins from mouse liver

Pyrene, a structural analog of benzo[a]pyrene, is an effective competing ligand for high affinity carcinogen binding proteins in mouse liver. A pyrenederivatized Sepharose gel was prepared for affinity chromatography purification of these proteins, and adsorbs all detectable [ 3H]B[a]P-binding activity from hepatic cytosol with the adsorption of less than 1% of total protein. Specific carcinogen binding activity is recovered from pyrene-derivatized Sepharose columns with the enrichment of a 33 kDa polypeptide. This chromatograpy resin represents a major step in the isolation of these unusual receptor-like binding proteins for aromatic hydrocarbon carcinogens.

Use of 5′-UTP-agarose for ribonuclease affinity chromatography

The use of commercially available 5′-UTP-agarose as an affinity chromatography resin for RNase has been described. It was shown that at pH 5.3, 0.025 m piperazine-HCl buffer was effective for the adsorption of active RNase A and exhibited little nonbiospecific binding as has been shown earlier for SepharoseaPhpUp [Stewart, G. R., and Stevenson, K. J. (1973) Biochem. J. 135, 427–441]. Phosphate buffer at either pH 3.0 or 5.45 eluted essentially all of the RNase activity added to the column; however, pH 5.45 was slightly more efficient. Competitive elution experiments with 2′(3′)-UMP yielded a linear plot of 1 (V − V o vs [I]. From this plot K I and K IM were calculated to be 70 and 130 μ m, respectively. It is suggested that since this material is different from that which is used most often for RNase A affinity chromatography, it may prove useful for RNase binding studies.

Synthesis of affinity chromatography resins for the purification of brain glutamate decarboxylase

In the present paper we describe the synthesis of Sepharose-boundN-(5'-phosphopyridoxyl)-amino-oxyacetic acid,N-(5'-phosphopyridoxyl)-canaline, andN-(5'-phosphopyridoxyl)-α,γ-diaminobutyric acid (Seph-DAB-PLP), designed for binding specifically brain glutamate decarboxylase (GAD). The three Sepharose-N-(phosphopyridoxyl)-amino acids were capable of binding GAD from a mouse brain soluble preparation. The enzyme can be purified up to 25-fold in one step by affinity chromatography on Seph-DAB-PLP.