Showing papers on "Affinity chromatography published in 1987"
TL;DR: Using in vitro mutagenesis, it is found that deletion of 12 amino acids from this region of gp120 leads to a complete loss of binding and a single amino acid substitution in this region results in significantly decreased binding, suggesting that sequences within this region are directly involved in the binding of gp 120 to the CD4 receptor.
858 citations
TL;DR: It is concluded that receptor oligomerization is an intrinsic property of the occupied EGF receptor and that it may play a role in the activation of the kinase function and the subsequent transmembrane signaling process.
Abstract: Epidermal growth factor (EGF) receptor from A-431 cells was purified by affinity chromatography with monoclonal anti-receptor antibodies. The purified radiolabeled receptor was incubated with EGF and then analyzed by gel electrophoresis under nondenaturing conditions. In these gels, the EGF receptor migrates in two forms: a fast-migrating (low) form and an EGF-induced slow-migrating (high) form. On the basis of the various control and calibration experiments described, it is concluded that the low form represents the monomeric 170-kilodalton EGF receptor and the high form represents an EGF receptor dimer. The binding of EGF causes a rapid, temperature-sensitive dimerization of the EGF receptor. Receptor dimerization is fully reversible and involves saturable, noncovalent interactions that are stable at neutral pH and in nonionic detergents. Both the monomeric and dimeric forms of the receptor bind EGF and undergo self-phosphorylation. The dimeric form of the receptor may possess higher ligand binding affinity, and it seems to be phosphorylated earlier than the monomeric form following the addition of EGF and [gamma-32P]ATP. On the basis of these results, it is concluded that receptor oligomerization is an intrinsic property of the occupied EGF receptor and that it may play a role in the activation of the kinase function and the subsequent transmembrane signaling process.
615 citations
Journal Article•
TL;DR: It appears that this monoclonal antibody is reacting with an epitope that is usually masked by oligosaccharide moieties in normal cells but which is exposed, perhaps due to aberrant glycosylation, in malignant cells.
Abstract: A mucin molecule, which has a molecular weight of greater than 400,000 and which carries tumor associated epitopes recognized by monoclonal antibodies HMFG-1 and HMFG-2, has been purified from human skimmed milk by affinity chromatography followed by passage through a size exclusion column. While treatment of the mucin with hydrogen fluoride for 1 h at 4°C removed the peripheral oligosaccharides, treatment with HF for 3 h at room temperature removed all of its lectin binding ability and revealed a dominant polypeptide of about 68,000. This appears to be the size of the mucin core protein. Monoclonal antibodies have been developed that react with the stripped and partially stripped molecule but not with the intact mucin. From the initial screening on histological sections one of these antibodies, SM-3, reacts with 91% of breast carcinomas but shows little or no reactivity on benign mammary tumors, normal resting, pregnant, or lactating breast. It appears that this monoclonal antibody is reacting with an epitope that is usually masked by oligosaccharide moieties in normal cells but which is exposed, perhaps due to aberrant glycosylation, in malignant cells.
522 citations
TL;DR: A Mr 35,000 protein with follicle-stimulating hormone release-inhibitory activity was isolated from porcine ovarian follicular fluid by heparin-Sepharose affinity chromatography, gel filtration on Sephacryl S-200, and multiple steps of high-performance liquid chromatography to signify its structural difference from inhibin.
Abstract: A Mr 35,000 protein with follicle-stimulating hormone release-inhibitory activity was isolated from porcine ovarian follicular fluid by heparin-Sepharose affinity chromatography, gel filtration on Sephacryl S-200, and multiple steps of high-performance liquid chromatography. The isolated molecule is highly enriched in cysteines and is composed of a single polypeptide chain. In addition, it has no sequence homology with the previously characterized follicular fluid inhibins, which are heterodimeric proteins of Mr 32,000 with follicle-stimulating hormone release-inhibiting activity. This protein specifically inhibits the basal secretion of follicle-stimulating hormone, but not that of luteinizing hormone, in the rat anterior pituitary monolayer culture system with a half-maximal effective dose of 2.5-6.0 ng/ml. Another form of the molecule of Mr 32,000 present in much lower concentration in follicular fluid was also isolated. It may differ from the Mr 35,000 form in glycosylation or carboxyl-terminal truncation. We suggest that this compound be called "follistatin" to signify its structural difference from inhibin.
457 citations
TL;DR: The purification steps include affinity chromatography on heparin-Sepharose, hydroxyapatite chromatography, gel filtration, and C18 reverse-phase HPLC, and the purified protein in conjunction with insoluble collagenous bone matrix induced new bone differentiation in vivo.
Abstract: Implantation of demineralized diaphyseal bone matrix in subcutaneous sites induces a sequence of events resulting in the local differentiation of endochondral bone. Demineralized bovine bone matrix was dissociatively extracted in 4.0 M guanidine hydrochloride and the bone-inductive proteins were purified greater than 12,000-fold. The purification steps include affinity chromatography on heparin-Sepharose, hydroxyapatite chromatography, gel filtration, and C18 reverse-phase HPLC. Since the purified protein in conjunction with insoluble collagenous bone matrix induced new bone differentiation in vivo we have designated this component osteogenin. The osteogenic potential is specific for osteogenin and is not exhibited by previously isolated growth factors.
385 citations
TL;DR: The systems described serve as a fine illustration for the biological role of lectin‐carbohydrate interactions and will lead to a deeper understanding of the molecular basis of infectious diseases, and perhaps also to new approaches for their prevention.
364 citations
TL;DR: A short DNA sequence element, the serum response element (SRE), which binds a nuclear protein, serum response factor (SRF), mediates transient transcriptional activation of c‐fos and cytoskeletal actin genes in response to serum factors.
Abstract: A short DNA sequence element, the serum response element (SRE), which binds a nuclear protein, serum response factor (SRF), mediates transient transcriptional activation of c-fos and cytoskeletal actin genes in response to serum factors. Variant SRE sequences with different affinities for HeLa cell SRF were synthesised. Binding of SRF to these sites in vitro correlates with the transcriptional properties of these elements in vivo, suggesting that SRF is a positively acting transcription factor. A 67-kd polypeptide was identified as the DNA-binding component of SRF by photoactivated DNA-protein cross-linking in vitro. The high affinity SRF-binding site was used to purify this polypeptide to virtual homogeneity in a single DNA affinity chromatography step.
338 citations
TL;DR: Drosophila heat shock activator protein, a rare transacting factor which is induced upon heat shock to bind specifically to the heat shock regulatory sequence in vivo, has been purified from shocked cells to more than 95 percent homogeneity by sequence-specific duplex oligonucleotide affinity chromatography.
Abstract: Drosophila heat shock activator protein, a rare transacting factor which is induced upon heat shock to bind specifically to the heat shock regulatory sequence in vivo, has been purified from shocked cells to more than 95 percent homogeneity by sequence-specific duplex oligonucleotide affinity chromatography. The purified protein has a relative molecular mass of 110 kilodaltons, binds to the regulatory sequence with great affinity and specificity, and strongly stimulates transcription of the Drosophila hsp70 gene. Studies with this regulatory protein should lead to an understanding of the biochemical pathway underlying the heat shock phenomenon.
336 citations
TL;DR: The relative affinities of the protein for a range of variant HSE sequences correlates with the ability of these sequences to support heat‐inducible transcription in vivo, suggesting that this polypeptide is involved in the activation of heat‐shock promoters.
Abstract: The promoters of heat shock genes are activated when cells are stressed. Activation is dependent on a specific DNA sequence, the heat-shock element (HSE). We describe the purification to homogeneity of an HSE-binding protein from yeast (Saccharomyces cerevisiae), using sequential chromatography of whole cell extracts on heparin-agarose, calf thymus DNA-Sepharose and an affinity column consisting of a repetitive synthetic HSE sequence coupled to Sepharose. The protein runs as a closely spaced doublet of approximately 150 kd on SDS-polyacrylamide gels; mild proteolysis generates a stable 70-kd fragment which retains DNA binding activity. The relative affinities of the protein for a range of variant HSE sequences correlates with the ability of these sequences to support heat-inducible transcription in vivo, suggesting that this polypeptide is involved in the activation of heat-shock promoters. However, the protein was purified from unshocked yeast, and may therefore represent an unactivated form of heat-shock transcription factor. Study of the purified protein should help to define the mechanistic basis of the heat-shock response.
314 citations
TL;DR: Recombinant human SP 28-36 isolated from the media of Chinese hamster ovary cells, transfected with a DNA construct encoding SP28-36, has similar carbohydrate-binding activity to the native proteins.
278 citations
TL;DR: The region of the fnbp‐gene encoding the fibronectin‐binding activity has been identified and subcloned in an expression vector based on the staphylococcal protein A gene and the resulting product in E. coli is an extracellular fusion protein consisting of two IgG‐binding domains of protein A followed by a fibronECTin‐ binding region.
Abstract: The gene encoding the fibronectin-binding protein (FNBP) from Staphylococcus aureus strain 8325-4 was isolated from a gene bank in pBR322. The original clone, containing a 6.5-kb insert, gave a functional product present in the periplasm of Escherichia coli. Analysis of polypeptides isolated after affinity chromatography on fibronectin-Sepharose followed by ion-exchange chromatography revealed two gene products, 87 and 165 kd in mol. wt. The amino acid compositions of these two polypeptides and a native FNBP from S. aureus strain Newman were very similar. Antibodies raised against the native FNBP from strain Newman precipitated the 125I-labelled 165-kd polypeptide, and unlabeled 165- and 87-kd polypeptides as well as native FNBP inhibited the immunoprecipitation reactions. The region of the fnbp-gene encoding the fibronectin-binding activity has been identified and subcloned in an expression vector based on the staphylococcal protein A gene. The resulting product in E. coli is an extracellular fusion protein consisting of two IgG-binding domains of protein A followed by a fibronectin-binding region. The fusion protein binds to fibronectin and completely inhibits the binding of fibronectin to intact cells of S. aureus.
TL;DR: The purified chimeric toxin is shown to selectively inhibit protein synthesis in IL-2 receptor bearing targeted cells, whereas cell lines which do not express the IL- 2 receptor are resistant to IL-1-toxin action.
Abstract: We have genetically replaced the diphtheria toxin receptor binding domain with a synthetic gene encoding interleukin-2 (IL-2) and a translational stop signal. The diphtheria toxin-related T-cell growth factor fusion gene encodes a 70 586-d polypeptide, pro-IL-2-toxin. The mature form of IL-2-toxin has a deduced mol. wt of 68,086 and is shown to be exported to the periplasmic compartment of Escherichia coli (pABI508), and contain immunologic determinants intrinsic to both its diphtheria toxin and IL-2 components. IL-2-toxin has been purified from periplasmic extracts of recombinant strains of E. coli (pABI508) by immunoaffinity chromatography using immobilized anti-IL-2. The purified chimeric toxin is shown to selectively inhibit protein synthesis in IL-2 receptor bearing targeted cells, whereas cell lines which do not express the IL-2 receptor are resistant to IL-2-toxin action.
TL;DR: Human class II histocompatibility antigens were purified from the Epstein-Barr virus-transformed human B lymphoblastoid cell line LG-2 by immunoaffinity chromatography for the first time all three subsets have been prepared as nonradioactive materials on a milligram scale.
TL;DR: SDM studies which test the role of electrostatics in antigen–antibody interactions are reported, finding that removal of two charged residues at the periphery of the combining site increases the affinity of the antibody for its antigen over 8-fold and decreases its ability to cross-react with closely-related antigens.
Abstract: A detailed description of the interactions between an antibody and its epitope is necessary to allow an understanding of the way in which antibodies bind to antigenic surfaces presented by foreign molecules. Ideally this should be done by analysis of crystal structures of antibody-antigen complexes, but so far only two of these are available. An alternative strategy combines molecular modelling with site-directed mutagenesis (SDM) and using this we have generated a preliminary model of the complex between Gloop2, an antibody raised against a peptide containing the 'loop' determinant of hen egg-white lysozyme (HEL) which also binds the native protein, and its epitope on the protein surface. The main predictions from our model were; (1) that the surface of interaction between the antibody and the antigen is large (20 A X 15 A) and involves all the complementarity-determining regions (CDRs), (2) that electrostatic interactions were important in the formation of the complex, and (3) that conformational changes in either the loop or in the CDRs may occur during the formation of the complex. Here we report SDM studies which test some of these predictions; removal of two charged residues at the periphery of the combining site increases the affinity of the antibody for its antigen over 8-fold and decreases its ability to cross-react with closely-related antigens. This result is at variance with our original prediction but can be accommodated within our newly refined model; the role of electrostatics in antigen-antibody interactions is now questionable.
TL;DR: Results indicate that the guanylate cyclase-coupled ANF receptor exists in a 180-kilodalton protein of rat adrenocortical carcinoma and represent a step toward the elucidation of the basic mechanism of cyclic GMP-mediated transmembrane signal transduction in response to a hormone.
Abstract: Atrial natriuretic factor (ANF) is a peptide hormone that is released from atria and regulates a number of physiological processes, including steroidogenesis in adrenal cortex and testes. The parallel stimulation of membrane guanylate cyclase and corticosterone production in isolated fasciculata cells of rat adrenal cortex has supported the hypothesis of a mediatory role for cyclic guanosine monophosphate (cyclic GMP) in signal transduction. A novel particulate guanylate cyclase tightly coupled with ANF receptor was purified approximately 273,000-fold by two-step affinity chromatography. The enzyme had a molecular size of 180 kilodaltons and was acidic in nature with a pI of 4.7. Its specific activity was 1800 nanomoles of cyclic GMP formed per minute per milligram of protein. The purified enzyme bound ANF with a specific binding activity of 4.01 nanomoles per milligram of protein, a value that is close to the theoretical binding activity of 5.55 nanomoles per milligram of protein for 1 mole of the ligand binding 1 mole of the receptor protein. These results indicate that the guanylate cyclase-coupled ANF receptor exists in a 180-kilodalton protein of rat adrenocortical carcinoma and represent a step toward the elucidation of the basic mechanism of cyclic GMP-mediated transmembrane signal transduction in response to a hormone.
TL;DR: The results of this study indicate that site E and site F, either singly or in combination, might constitute the receptor binding domain of apoB-100.
TL;DR: The affinity purified kidney enzyme has similarities to but also significant differences in structural and enzymatic properties from those reported for other vacuolar H+ATPase.
TL;DR: Results indicate that the Mr 93,000 polypeptide is a peripheral membrane protein that is located at the cytoplasmic face of the postsynaptic glycine receptor complex.
Abstract: The glycine receptor of mammalian spinal cord is an oligomeric membrane protein that, after affinity purification on aminostrychnine-agarose or immobilized antibody, contains three polypeptides of Mr 48,000, 58,000, and 93,000. Here, the association and the properties of the polypeptides of the rat glycine receptor were investigated. Upon phase partitioning in the nonionic detergent Triton X-114, the three receptor polypeptides behaved as a hydrophilic protein complex exhibiting phospholipid binding. Sucrose gradient centrifugation or gel filtration in the presence of dithiothreitol and Triton X-100 separated the Mr 93,000 polypeptide from the Mr 48,000 and 58,000 polypeptides, which harbor the antagonist binding site of the glycine receptor. Alkaline or dimethylmaleic acid anhydride treatment of crude synaptic membrane fractions resulted in extraction of the Mr 93,000 polypeptide. Lectin binding was observed for the Mr 48,000 and 58,000 glycine receptor subunits but not the Mr 93,000 polypeptide. These results indicate that the Mr 93,000 polypeptide is a peripheral membrane protein that is located at the cytoplasmic face of the postsynaptic glycine receptor complex.
TL;DR: Analysis of the binding of the 29-kDa amino-terminal fibronectin fragment to the 210-k da receptor adsorbed in microtiter wells suggests that one receptor molecule can bind six to nine fibronECTin molecules, which suggests that the large receptor protein contains several binding sites for fibronctin.
TL;DR: The purification and characterization of acetylcholinesterase from heads of the fruit fly Drosophila and the hydrolyzed butyrylthiocholine much more efficiently than these eel or human enzymes are described, an indication that the fly head enzyme has a substrate specificity intermediate between mammalian acetyl Cholinestersterases and butyRYlchlinesterases.
TL;DR: Quantitative gel slicing of 3H- and 125I-labeled nonreduced enzyme supported a structural model in which the tetrameric enzyme is a dimer of nonidentical catalytic subunit dimers, one of which involves a direct intersubunit disulfide linkage between two 70-kDa catalyticSubunit monomers and the second of which contains two disulfides linkages through an intervening 125I
TL;DR: The biotin-binding properties of the nonglycosylated avidin were equivalent to those obtained for the native (gly cosylated) avidin molecule, indicating that the oligosaccharide moiety is not essential for the binding activity.
Abstract: A commercially available, purified preparation of avidin was found to comprise two polypeptide bands (Mr 18,000 and Mr 15,500 respectively). Both bands bound biotin as assessed by biotin overlays of protein blots. The Mr 15,500 polypeptide was found to differ from the Mr 18,000 polypeptide only in its sugar content. When the commercial preparation was applied to a concanavalin A affinity column, the glycosylated forms were retarded as expected, and homotypic nonglycosylated avidin tetramers which failed to bind selectively to the column were collected in the effluent. The biotin-binding properties of the nonglycosylated avidin were equivalent to those obtained for the native (glycosylated) avidin molecule, indicating that the oligosaccharide moiety is not essential for the binding activity.
TL;DR: Purity and yields as judged by gel electrophoresis and densitometric scanning of the B protein were approximately 1.7-fold higher due to partial loss in hormone binding activity at the elution step and these new MAbs are valuable reagents for further studies of human receptor structure and function and for clinical immunodetection of PR in breast tumors.
Abstract: A monoclonal antibody (MAb), designated PR-6, produced against chick oviduct progesterone receptors [Sullivan, W. P., Beito, T. G., Proper, J., Krco, C. J., & Toft, D. O. (1986) Endocrinology (Baltimore) 119, 1549-1557] cross-reacts with the Mr 120,000 human B receptors. An immunomatrix prepared with PR-6 was used to purify progesterone receptors (PR) from T47D human breast cancer cells. Single-step immunoaffinity chromatography results in enrichment of B receptors (identified by immunoblot with PR-6 and by photoaffinity labeling with [3H]promegestone) to a specific activity of 1915 pmol/mg of protein (or 23% purity) and with 27% yield. Purity and yields as judged by gel electrophoresis and densitometric scanning of the B protein were approximately 1.7-fold higher due to partial loss in hormone binding activity at the elution step. A second purification step by diethylaminoethyl chromatography gives further enrichment to 3720 pmol/mg of protein (or 44% purity) to yield essentially two proteins, 120-kilodalton (kDa) B receptors and a 76-kDa non-steroid binding protein, each in approximately equivalent amounts. B receptors purified under these conditions are transformed and biologically active. They were maintained as undegraded 120-kDa doublets and retained both hormone and DNA binding activities. Isolated B receptors were free of the 90-kDa non-steroid binding protein observed to be associated with 8S untransformed receptors in other systems and were free also of the non-hormone binding 105-108-kDa B antigen described previously to copurify with chick PR. These purified B receptors were used as immunogen for production of four monoclonal antibodies against human PR. Three of the MAbs, designated as B-30 (IgG1), B-64 (IgG1), and B-11 (IgM), are specific for B receptors. The fourth MAb, A/B-52 (IgG1), reacts with both A and B receptors. The IgG MAbs are monospecific for human PR since they recognize and absorb native receptor-hormone complexes, displace the sedimentation of 4S receptors on salt containing sucrose gradients, and, by immunoblot assay of crude T47D cytosol, react only with receptor polypeptides. Although mice were injected with B receptors only, production of A/B-52 which recognized both A and B receptors provides evidence that these two proteins share regions of structural homology. These new MAbs are valuable reagents for further studies of human receptor structure and function and for clinical immunodetection of PR in breast tumors.
TL;DR: Several fusions between the gene for human insulin-like growth factor I (IGF-I) and the genes for different IgG-binding fragments of staphylococcal protein A were assembled and compared regarding expression, secretion, and purification of the peptide hormone.
Abstract: Several fusions between the gene for human insulin-like growth factor I (IGF-I) and the genes for different IgG-binding fragments of staphylococcal protein A were assembled and compared regarding expression, secretion, and purification of the peptide hormone. After IgG affinity purification of the fusion proteins from the growth medium of Staphylococcus aureus or Escherichia coli, native IGF-I was released by cleavage of an Asn-Gly peptide bond with hydroxylamine. An optimized expression system based on a modified synthetic IgG-binding domain (z), resistant to hydroxylamine, gave the highest yield of fusion protein. After cleavage, the hormone could be separated from the IgG-binding moiety and from noncleaved fusion protein by a second passage through the IgG affinity column. The biological activity and the purity of the IGF-I obtained were confirmed by a radioreceptor assay, N-terminal sequence analysis, polyacrylamide gel electrophoresis, isoelectric focusing, and high-performance liquid chromatography.
TL;DR: The analysis of seminiferous tubules indicates that the technique may be of value for the identification of novel subunits in rat tissue extracts and preliminary separations of subunits from human GSH transferases are described.
Abstract: A simple method is described for the separation and quantification of the subunits of GSH transferases present in rat tissue extracts. This method, involving GSH-agarose affinity chromatography followed by reverse-phase h.p.l.c., is rapid and sufficiently sensitive to measure 5 micrograms of each subunit in a mixture. Examples are given of its application to extracts of rat kidney, adrenal, testicular interstitial cells and seminiferous tubules. The analysis of seminiferous tubules indicates that the technique may be of value for the identification of novel subunits. Preliminary separations of subunits from human GSH transferases are also described.
TL;DR: It is concluded that the 28K IGF-binding protein from amniotic fluid, like the previously purified 53K binding protein, has high affinity for both IGF-I and IGF-II, that it coexists in amniotics fluid with BP-53 or a related protein, and that the levels of both proteins decline with increasing fetal maturity.
Abstract: A binding protein for insulin-like growth factors (IGFs) has been purified from human amniotic fluid by IGF-I affinity chromatography and high performance reverse phase chromatography. This protein, with an apparent molecular mass of 28K nonreduced and 34K reduced, had an identical amino-terminus to a previously purified binding protein from amniotic fluid and to placental protein 12. The purified preparation (BP-28) bound both IGFs with high affinity [Ka, 6.55 +/- 2.24 (+/- SD) L/nmol for IGF-I and 3.23 +/- 1.05 L/nmol for IGF-II], with approximately 0.5 mol binding sites/mol BP-28 for either ligand. A 53K IGF-binding protein purified from human plasma (BP-53) did not cross-react in a RIA for BP-28, and BP-28 had less than 0.1% molar cross-reactivity in a RIA for BP-53. Human amniotic fluid reacted strongly in both assays. Fractionation of amniotic fluid samples by reverse phase chromatography showed that BP-28 and BP-53 immunoreactivities were present on separate proteins. In 40 third trimester amniotic fluid samples selected to cover a wide range of lecithin to sphingomyelin ratios, the mean concentrations of BP-28 and BP-53 were 37.6 +/- 17.6 (+/- SD) and 4.6 +/- 1.6 mg/L, respectively. Significant negative correlations were found between the levels of both BP-28 and BP-53 and the lecithin to sphingomyelin ratio, suggesting an association between the levels of both proteins and the degree of fetal maturity. A significant positive association was also found between the levels of BP-28 and BP-53. We conclude that the 28K IGF-binding protein from amniotic fluid, like the previously purified 53K binding protein, has high affinity for both IGF-I and IGF-II, that it coexists in amniotic fluid with BP-53 or a related protein, and that the levels of both proteins decline with increasing fetal maturity.
TL;DR: A streptavidin/biotin-based immunoaffinity system was optimized to isolate herpesvirus (human cytomegalovirus) immediate early proteins or late glycoproteins from crude infected cell lysates.
TL;DR: Gel retardation and footprint analysis have shown that purified KBF1 has a binding activity specific for both H‐2 and beta 2‐microglobulin enhancer sequences, suggesting that the expression of both H•2 and Beta 2‐ microglobulins genes utilizes a common regulatory mechanism.
Abstract: An enhancer binding factor, designated KBF1, has been purified from the nuclear extract of mouse BW5147 thymoma cells by five column chromatography steps including a sequence-specific DNA affinity column. Gel retardation and footprint analysis have shown that purified KBF1 has a binding activity specific for both H-2 and beta 2-microglobulin enhancer sequences. After SDS-polyacrylamide gel electrophoresis of the most purified preparation a 48-kd protein showed, after elution and renaturation, a binding activity to both enhancer sequences. These findings suggest that the expression of both H-2 and beta 2-microglobulin genes utilizes a common regulatory mechanism.
TL;DR: In this article, neutralizing monoclonal antibodies were used as immunoadsorbents to purify recombinant TNF-α and TNFβ from E. coli lysates.
Abstract: Monoclonal antibodies were produced against two structurally related tumor necrosis factors (TNFs), TNF -α (previously called tumor necrosis factor) and TNF-β (previously called lymphotoxin). The potential of these antibodies for the purification of TNFs, the development of specific immunoassays, and for defining the antigenic and functional domains of these cytokines was investigated. None of the monoclonal antibodies cross-reacted with both TNF-α and TNF-β, or reacted with synthetic peptides which represented several of the regions of homology between these cytokines. Neutralizing monoclonal antibodies were utilized as immunoadsorbents to purify recombinant TNF-α and TNF-β from E. coli lysates. TNFs purified by this method were greater than 98 percent pure by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and exhibited specific activities that were the same as TNFs isolated from natural sources using conventional Chromatographic techniques. In addition, specific ELISA assays were developed th...
TL;DR: It is demonstrated that the expression system can be used for the production of biologically active human insulin–like growth factor I in a 1000 litre scale.
Abstract: A genetic approach to facilitate large–scale production and down stream processing of heterologous proteins expressed in bacteria is described. The gene is fused after a synthetic fragment encoding two IgG–binding domains derived from staphylococcal protein A. The fusion product is secreted to the growth medium of Escherichia coli, and purified using the IgG–binding moiety as affinity “tail”. We demonstrate that the expression system can be used for the production of biologically active human insulin–like growth factor I in a 1000 litre scale. The process includes fermentation, cell separation, affinity chromatography on IgG Sepharose Fast Flow and site specific chemical cleavage of the fusion protein.