A physicochemical study of protein G, a molecule with unique immunoglobulin G-binding properties

Protein G, an IgG-binding molecule, was prepared from the cell walls of a group G streptococcal strain, G-148. The protein could be extracted from the cells by papain digestion and purified by the sequential use of ion-exchange chromatography on DEAE-Sephadex, affinity chromatography on Sepharose-coupled human IgG, and gel chromatography on Sephadex G-200. Two protein bands with similar molecular weight, 34,000 and 36,000, were obtained when analyzing the pure protein G on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The yield using this purification scheme was 27% of the protein G solubilized from the cells or 70 micrograms/ml packed bacteria. The Stokes radius and frictional ratio of protein G were determined to 3.53 nm and 1.64, respectively, suggesting an elongated fibrous molecule. The protein did not contain any intrachain disulfide bonds. The amino acid composition of protein G was determined and was found to be different from that of protein A, the well known staphylococcal IgG-binding protein. The equilibrium constants of the reactions between protein G and human, rabbit, mouse, and goat polyclonal IgG, determined by Scatchard plots, ranged between 1 X 10(10) and 7 X 10(10), for rat polyclonal IgG 1.4 X 10(9), and human monoclonal IgG1, IgG2, IgG3, and IgG4 between 2 X 10(9) and 6 X 10(9). These affinity constants were always greater than the corresponding values for protein A. The binding between protein G and various polyclonal and monoclonal IgG was pH dependent between 2.8 and 10, strongest at pH 4 and 5, and weakest at pH 10.     

Isolation of a low molecular mass vasoactive intestinal peptide binding protein.

A vasoactive intestinal peptide (VIP) binding protein was purified in active form by detergent solubilization of lung membranes, gel filtration, VIP-Sepharose affinity chromatography, reverse phase high performance liquid chromatography, and anion exchange chromatography. The mass of this protein was estimated at 18 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 17 kDa by gel filtration. The binding of VIP by this protein was inhibited by Mg2+, covalent cross-linking of [Tyr10-125I]VIP to the protein produced two radioactive bands at 22 and 26 kDa identified by electrophoresis, and the purified protein exhibited saturable and high affinity binding of VIP and the related neuropeptide, rat growth hormone releasing factor.     

Purification of human lymphoblastoid interferon by a simple procedure with high yields

Human Namalwa cell interferon, induced by Sendai virus and composed of a single species with molecular weight of 17,000, was purified to 4.5 X 10(8) international reference units/mg of protein by a combination of salt precipitation, ion exchange chromatography, metal chelate chromatography, hydrophobic chromatography, and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. By immunization of a rabbit with this purified interferon and by extensive absorption with Namalwa cells and an impurity column, highly specific antibody was obtained. Namalwa cells, treated with 5-bromo-2'-deoxyuridine, produced 10-fold more interferon upon induction by Sendai virus. Interferon in this case consisted of heterogeneous species with molecular weight ranging from 15,000 to 24,000. These heterogeneous interferon molecules were purified to 7.6 X 10(8) international reference units/mg of protein by successive chromatography using immobilized highly specific rabbit anti-interferon antibody, Blue Sepharose, and immobilized goat anti-rabbit IgG antibody. The overall recovery of interferon activity was 72%, and the purity of the final preparation was ascertained by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.     

Characterization of a pathogenesis-related class 10 protein (PR-10) from Astragalus mongholicus with ribonuclease activity.

A pathogenesis-related (PR) class 10 protein (designated AmPR-10) was first isolated from the Chinese medicinal material Astragalus mongholicus using a combination of affinity chromatography on Zn-chelate Agarose 4B, ion exchange chromatography on QAE Sephadex A-25 and gel filtration on Sephadex G50. The purified AmPR-10 showed a single band with a molecular mass of 17.2kDa in SDS-PAGE. The molecular mass of intact AmPR-10 was determined to be 32.8kDa by gel filtration. Thus, AmPR-10 is a dimeric protein composed of two identical subunits. AmPR-10 was a glycoprotein detected by periodic acid-Schiff (PAS) staining and its neutral carbohydrate content was 13.7%. The carbohydrate was mainly composed of 73.0% (w/w) arabinose, 15.0% (w/w) glucose and 4.8% (w/w) fructose on the basis of high-performance anion exchange chromatography (HPAEC) analysis. Its N-terminal sequence of 15 amino acid residues was determined as GVISFNEETISTVAP, and showed significant sequence homology to some pathogenesis-related (PR) class 10 proteins. This sequence had 80% identity with the PR-10 protein LlPR10.1C from Lupinus luteus (yellow lupine) followed by 73.3% identity with the PR-10 protein PR10.2 from Medicago sativa (alfalfa), suggesting it is a new member of PR-10 proteins. AmPR-10 exhibited ribonuclease (RNase) activity as do some other PR-10 proteins. The optimal pH and temperature for RNase activity were pH 6.0 and 60 degrees C, respectively. The RNase activity was stable within pH 5.0-11.0. It was stable up to 60 degrees C at pH 6.0. The purification and characterization of AmPR-10 in this investigation furnish additional data to the relatively scanty literature pertaining to Astragali radix proteins.     

Purification of boiling-soluble antifreeze protein from the legume Ammopiptanthus mongolicus

A boiling-soluble antifreeze protein (AFP) was purified from the winter leaves of Ammopiptanthus mongolicus an evergreen legume species surviving in the cold desert of northwest of China. Purification was achieved by using a procedure consisting of a heat treatment step followed by consecutive chromatography, including ion-exchange chromatography (DEAE-Cellulose 52, Source 15Q), molecular exclusion chromatography with Sephacryl S300, and hydrophobic interaction chromatography (Poros 20HP2). This AFP showed thermal hysteresis activity and could modify the normal growth of ice crystals. The thermal hysteresis activity (THA) of this purified antifreeze protein is 0.15 degrees C at a concentration of 10 mg/mL, and its molecular mass is approximately 28 kD by SDS-PAGE analysis.     

Rhodanese from Cercopithecus aethiops (vervet monkey) liver. I. Purification and some physical characteristics

Rhodanese (thiosulphate sulphurtransferase , EC 2.8.1.1.) from Cercopithecus aethiops (vervet monkey) liver has been isolated and purified by means of extraction, ammoniumsulphate and pH fractionation, anion-exchange chromatography, Sephacryl S-300 gel chromatography and cation-exchange chromatography. A yield of about 10% pure enzyme with a specific activity of 242 U/mg protein corresponding to a purification factor of 523 was obtained. The enzyme was physically characterized and its homogeneity determined by electrophoretic studies and gel chromatography. The rhodanese enzyme has a molecular weight of 37,000 daltons, a D020 ,w value of 7.6 X 10(-7) cm2 sec-1, a Stokes radius (molecular size) of 2.75 X 10(-7) cm and a frictional ratio of 1.071.     

Characterization of an antibiotic produced by Alteromonas luteoviolacea Gauthier 1982,85 isolated from Kinko Bay, Japan

An antibiotic produced by Alteromonas luteoviolacea strain 9K-V10 was recovered after cold acetone precipitation of culture supernatant fluids or lysates that had beenfrozen and thawed. The precipitate obtained from cell-free lysates was fractionated by DEAE ion-exchange chromatography. Further purification by gel-filtration chromatography yielded a single peak of antibiotic activity that corresponded to a protein peak with a molecular mass of approximately 100 kDa. After non-denaturing polyacrylamide gel electrophoresis, antibiotic activity co-migrated with a protein band. The isoelectric point of the antibiotic was estimated to be 7·7. Treatment of the concentrated active fraction with proteinase K or heating at 70°C for 10 min resulted in total loss of antibiotic activity. These results show that the antibiotic produced by Alt. luteoviolacea 9K-V10 is of a proteinaceous nature.     

Characterization of the haloacid dehalogenase from Xanthobacter autotrophicus GJ10 and sequencing of the dhlB gene.

The haloacid dehalogenase of the 1,2-dichloroethane-utilizing bacterium Xanthobacter autotrophicus GJ10 was purified from a mutant with an eightfold increase in expression of the enzyme. The mutant was obtained by selecting for enhanced resistance to monobromoacetate. The enzyme was purified through (NH4)2SO4 fractionation, DEAE-cellulose chromatography, and hydroxylapatite chromatography. The molecular mass of the protein was 28 kDa as determined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 36 kDa as determined with gel filtration on Superose 12 fast protein liquid chromatography. The enzyme was active with 2-halogenated carboxylic acids and converted only the L-isomer of 2-chloropropionic acid with inversion of configuration to produce D-lactate. The activity of the enzyme was not readily influenced by thiol reagents. The gene encoding the haloacid dehalogenase (dhlB) was cloned and could be allocated to a 6.5-kb EcoRI-BglII fragment. Part of this fragment was sequenced, and the dhlB open reading frame was identified by comparison with the N-terminal amino acid sequence of the protein. The gene was found to encode a protein of 27,433 Da that showed considerable homology (60.5 and 61.0% similarity) with the two other haloacid dehalogenases sequenced to date but not with the haloalkane dehalogenase from X. autotrophicus GJ10.     

Purification of a novel UV-damaged-DNA binding protein highly specific for (6-4) photoproduct.

UV damage-specific binding proteins are considered to play important roles in early responses of cells irradiated with UV, including damage recognition in the DNA repair process. We have surveyed nuclear and cytoplasmic proteins which bind selectively to UV-irradiated DNA using an electrophoretic mobility shift assay. We detected four distinct binding activities with different mobilities in fractions separated from HeLa cells by heparin chromatography. Three of them were found in nuclear extracts and one in cytoplasmic extracts. We purified one of the binding factors from nuclear extracts to homogeneity, which was designated NF-10 (the 10th fraction of nuclear extract on heparin chromatography). It migrated as a 40 kDa polypeptide in SDS-PAGE, and bound to UV-irradiated double- stranded DNA but not to unirradiated DNA. The binding pattern of the NF-10 protein to DNA irradiated with UV corresponded to the induction kinetics of (6-4) photoproduct. Removal of (6-4) photoproducts from UV- irradiated DNA by (6-4) photoproduct-specific photolyase diminished the binding of NF-10 protein. These results suggest that the NF-10 protein binds to UV-damaged DNA through (6-4) photoproduct. Immunoblot analysis using a monoclonal antibody revealed that the NF-10 protein was expressed in cell lines from all complementation groups of xeroderma pigmentosum, indicating that the NF-10 protein is a novel UV-damaged-DNA binding protein.     

Preparative parallel protein purification (P4)

In state of the art drug discovery, it is essential to gain structural information of pharmacologically relevant proteins. Increasing the output of novel protein structures requires improved preparative methods for high throughput (HT) protein purification. Currently, most HT platforms are limited to small-scale and available technology for increasing throughput at larger scales is scarce. We have adapted a 10-channel parallel flash chromatography system for protein purification applications. The system enables us to perform 10 different purifications in parallel with individual gradients and UV monitoring. Typical protein purification applications were set up. Methods for ion exchange chromatography were developed for different sample proteins and columns. Affinity chromatography was optimized for His-tagged proteins using metal chelating media and buffer exchange by gel filtration was also tested. The results from the present system were comparable, with respect to resolution and reproducibility, with those from control experiments on an AKTA purifier system. Finally, lysates from 10 E. coli cultures expressing different His-tagged proteins were subjected to a three-step parallel purification procedure, combining the above-mentioned procedures. Nine proteins were successfully purified whereas one failed probably due to lack of expression.     

A protein with inhibitory activity on cell-free translation from cultured mycelia of the edible mushroom Tricholoma lobayense

Cultured mycelia of the edible mushroom Tricholoma lobayense were extracted with cold saline. Proteins were precipitated from the extract by addition of (NH4)2SO4. The precipitate was dissolved and dialyzed before ion exchange chromatography on DEAE-cellulose. Ability to inhibit translation in a rabbit reticulocyte lysate was located in the unadsorbed fraction which was then subjected to affinity chromatography on Affi-gel Blue gel. The strongest activity was again retained by the unadsorbed fraction. Ion exchange chromatography on CM-cellulose resulted in fractionation of this fraction into an unadsorbed and two adsorbed peaks. Cell-free translation inhibitory activity was concentrated in the fraction eluted with 100 mM NaCl in 10 mM NH4OAc (pH 5.4). The translation-inhibitory protein possessed a molecular weight of 30 kDa as estimated by gel filtration using a fast protein liquid chromatography system and sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Purification of rat chondrosarcoma xylosyltransferase

The chondroitin sulfate chain-initiating enzyme, UDP-d-xylose:core protein β-d-xylosyltransferase has been purified over 600-fold from the high-speed supernatant fraction of a rat chondrosarcoma. The purification procedure involved differential centrifugation, gel chromatography on Sephadex G-200, and affinity chromatography on a matrix consisting of core protein bound to Sepharose. The purified enzyme was homogenous by electrophoretic and immunological criteria, had a molecular weight between 95,000 and 100,000 and contained approximately 10% carbohydrate. The K_m value for UDP-xylose was 1 × 10^?5, m and for the core-protein acceptor was 330 mg/liter.     

Purification of the c-fos enhancer-binding protein.

We have purified the c-fos enhancer-binding protein from HeLa cell nuclear extracts. The key purification steps involved chromatography on a nonspecific DNA affinity column, from which binding activity and other protein were eluted at low salt concentrations, followed by chromatography on a specific oligonucleotide affinity column, from which the enhancer binding activity was specifically eluted at high salt concentrations. The purified protein had a strong affinity for the c-fos enhancer dyad symmetry sequence, with an equilibrium dissociation constant of 3.3 x 10(-11) M. This affinity was at least 50,000-fold stronger than that found for nonspecific DNA sequences.     

Purification and characterization of the chaperonin 10 and chaperonin 60 proteins from Rhodobacter sphaeroides.

Two heat-shock proteins that show high identity with the Escherichia coli chaperonin 60 (groEL) and chaperonin 10 (groES) chaperonin proteins were purified and characterized from photolithoautotrophically grown Rhodobacter sphaeroides. The proteins were purified by using sucrose density gradient centrifugation and Mono-Q anion-exchange chromatography. In the presence of 1 mM ATP, the chaperonin 10 and chaperonin 60 proteins bound to each other and comigrated as a large complex during sucrose density gradient centrifugation. The native molecular weights of each protein as determined by gel filtration chromatography were 889,200 for chaperonin 60 and 60,000 for chaperonin 10. Chaperonin 60 is comprised of monomers with a molecular weight of 61,000 and chaperonin 10 is comprised of monomers with a molecular weight of 12,700 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Chaperonin 60 was 9.3% of the total soluble cell protein during photolithoautotrophic growth which increased to 28.5% following heat-shock treatment. When cells were grown photoheterotrophically or chemoheterotrophically, chaperonin 60 was reduced to 6.7% and 3.5%, respectively, of the total soluble protein. The N-terminal amino acid sequence of each protein was determined; chaperonin 60 of R. sphaeroides showed 72% identity to E. coli chaperonin 60 protein, and R. sphaeroides chaperonin 10 showed 45% identity with E. coli chaperonin 10. R. sphaeroides chaperonin 60 catalyzed ATP hydrolysis with a specific activity of 134 nmol min-1 mg-1 (kcat = 0.13 s-1) and was inhibited by R. sphaeroides chaperonin 10, but not E. coli chaperonin 10. The E. coli chaperonin 60 ATPase activity was inhibited by chaperonin 10 from both R. sphaeroides and E. coli.     

High level expression and purification of the Epstein-Barr virus encoded cytokine viral interleukin 10: efficient removal of endotoxin

To characterize the structural and functional properties of viral interleukin 10 (vIL-10), its cDNA was cloned into the bacterial expression vector pMAL-c2, which directs the synthesis of the inserted gene as a fusion protein with maltose binding protein (MBP). The MBP-vIL-10 fusion protein was expressed in Escherichia coli and purified from cell lysates using amylose resin chromatography. Viral interleukin 10 (IL-10) was released from the fusion protein by cleavage with the proteolytic enzyme factor Xa. We show that vIL-10 will bind to heparin and use this property to purify vIL-10 from factor Xa cleaved products and trace contaminants using heparin agarose chromatography. A simple one-step procedure is described for the removal of endotoxins from heavily contaminated vIL-10 preparations. The protocol exploits the high binding affinity of MBP for amylose resin or vIL-10 for heparin and the ability of Triton-X114 to dissociate endotoxins from proteins. The biological activity of purified vIL-10 was demonstrated through its ability to inhibit interferon gamma (IFN-gamma) production by mitogen activated peripheral blood mononuclear cells and to down-regulate HLA-class II expression on activated monocytes/macrophages. The availability of an efficient expression and purification strategy for vIL-10 together with appropriate assays will contribute to a greater understanding of how vIL-10 has evolved to retain and modify those activities of cellular IL-10 best suited for Epstein-Barr virus (EBV)'s specialized niche within the host.     

Purification and characterization of human IL-10/Fc fusion protein expressed in Pichia pastoris

Interleukin (IL)-10 is an anti-inflammatory cytokine that could be potentially applied for clinical therapy. However, its short circulating half-life in the serum limits its clinical applications. In this study, we designed a fusion protein containing human IL-10 and an IgG Fc fragment (hIL-10/Fc), and expressed it in Pichia pastoris. This hIL-10/Fc fusion protein was purified from the culture supernatant using MabSelect affinity chromatography and size-exclusion chromatography. The hIL-10/Fc yield was about 5mg/L in shake flasks, with purity exceeding 95%. In addition, the hIL-10/Fc fusion protein suppressed the phytohemagglutinin-induced IFN-γ production in human peripheral blood mononuclear cells. Pharmacokinetic study also revealed that hIL-10/Fc has a prolonged circulating half-life of about 30h in rats. More importantly, the hIL-10/Fc fusion protein displayed highly specific biological activity, which was slightly higher than that of the commercial recombinant human IL-10 (rhIL-10). Therefore, P. pastoris is useful in the large-scale production of hIL-10/Fc fusion protein for both research and therapeutic applications.     

Routine manufacture of recombinant proteins using expanded bed adsorption chromatography

Two different recombinant human proteins were purified directly from Pichia pastoris whole cell fermentation broth, containing 30–44% biomass (wet weight percent), by strong cation exchange expanded bed adsorption chromatography. Expanded bed adsorption chromatography provided clarification, product purification and product concentration in a single unit operation at large scale (2000-l nominal fermentation volume). The efficiency of expanded bed adsorption chromatography resulted in a short process time, high process yield, and limited proteolytic degradation of the target proteins. The separations were operated using a 60-cm (d) column run at 14 l/min. For one protein, expanded bed adsorption chromatography resulted in an average product recovery of 113% (relative to fermentation supernatant) and a purity of 89% (n=10). For the other protein, the average product recovery was 99% (relative to fermentation supernatant) and the purity was 62.1 (n=10). Laboratory experiments showed that biomass reduced product dynamic binding capacity for protein 2.     

Purification of a special estrogen-binding protein from the liver of rats by affinity chromatography on estradiol sepharose

A procedure has been developed for purification of a special rat liver estrogen-binding protein. It includes protein precipitation by ammonium sulfate, gel filtration, ion exchange chromatography, and affinity chromatography on estradiol sepharose. The protein is purified 2260-fold with a 27% yield. Upon electrophoresis in 10% PAG in the presence of sodium dodecylsulfate the protein gives one polypeptide strip with a molecular weight of 31.200 +/- 400 dalton.     

Purification of ferrichrome synthetase from Aspergillus quadricinctus and characterisation as a phosphopantetheine containing multienzyme complex

Aspergillus quadricinctus was grown under iron limitation to induce the enzymes for ferrichrome biosynthesis. The mycelium was disintegrated by ultraturrax homogenization, and ferrichrome synthetase was purified by column chromatography on DEAE cellulose, hydroxyapatite and Bio-Gel A-5m. The enzyme was almost homogeneous in single fractions as shown in gel electrophoresis under non-denaturating conditions. By fast-protein liquid chromatography on Superose 6, the purified ferrichrome synthetase (molecular weight 9.6.10(5) dissociated partly into an enzyme complex with reduced ferrichrome synthetase activity of 8 x 10(5) Da, one acetylhydroxyornithine (AHO) activating protein of 5.5 x 10(5) Da and one glycine activating protein of 4 x 10(5) Da. After SDS treatment the AHO activating protein dissociated into subunits of 9 x 10(4) Da, while the glycine activating protein dissociated into subunits of 5 x 10(4) Da and 4 x 10(4) Da in a molar ratio of 6:1. No subunits were found after SDS treatment of the larger of the two ferrichrome synthetizing enzyme complexes. Pantetheine was detected in protein bands of defined molecular weights (4 x 10(4), 9 x 10(4) and greater than 3.4 x 10(5) after SDS polyacrylamide gel electrophoresis. Gel slices were cut out, and the growth factor activity for Lactobacillus plantarum ATCC 8014 was analyzed. The calculated content was 2 mol of pantetheine per mol of ferrichrome synthetase of 9.6 x 10(5) Da.     

A Leucine Zipper-Like Domain Is Essential for Dimerization and Encapsidation of Bluetongue Virus Nucleocapsid Protein VP4

The bluetongue virus (BTV) minor protein VP4, with molecular mass of 76 kDa, is one of the seven structural proteins and is located within the inner capsid of the virion. The protein has a putative leucine zipper near the carboxy terminus of the protein. In this study, we have investigated the functional activity of this putative leucine zipper by a number of approaches. The putative leucine zipper region (amino acids [aa] 523 to 551) was expressed initially as a fusion protein by using the pMAL vector of Escherichia coli, which expresses a maltose binding monomeric protein. The expressed fusion protein was purified by affinity chromatography, and its size was determined by gel filtration chromatography. Proteins of two sizes, 51 and 110 kDa, were recovered, one equivalent to the monomeric form and the other equivalent to the dimeric form of the fusion protein. To prove that the VP4-derived sequence was responsible for dimerization of this protein, a mutated fusion protein was created in which a VP4 leucine residue (at aa 537) within the zipper was replaced by a proline residue. Analyses of the mutated protein demonstrated that the single mutation indeed prevented dimerisation of the protein. The dimeric nature of VP4 was further confirmed by using purified full-length BTV-10 VP4 recovered from recombinant baculovirus-expressing BTV-10 VP4-infected insect cells. Using chemical cross-linking and gel filtration chromatography, we documented that the native VP4 indeed exists as a dimer in solution. Subsequently, Leu537 was replaced by either a proline or an alanine residue and the full-length mutated VP4 was expressed in the baculovirus system. By sucrose density gradient centrifugation and gel filtration chromatography, these mutant forms of VP4 were shown to lack the ability to form dimers. The biological significance of the dimeric forms of VP4 was examined by using a functional assay system, in which the encapsidation activity of VP4 into core-like particles (CLPs) was studied (H. LeBlois, T. French, P. P. C. Mertens, J. N. Burroughs, and P. Roy, Virology 189:757–761, 1992). We demonstrated conclusively that dimerization of VP4 was essential for encapsidation by CLPs.