Rapid purification of protein kinase C by high performance liquid chromatography

Protein kinase C was purified from rat brain cytosol by using a high performance liquid chromatography (HPLC), Pharmacia FPLC system. This procedure employed a column chromatography on DE-52, followed by three steps of HPLC procedures with threonine-Sepharose (prepared as described in this report), TSK gel Phenyl-5PW (Toyo Soda), and TSK gel G3000SW (Toyo Soda) columns. Starting from about 30 g of rat brain, approximately 200 micrograms of pure enzyme was obtained. The procedure was very simple and highly reproducible. The enzyme thus obtained was nearly pure by silver staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the presence of 10% (w/v) glycerol and 0.05% (w/v) Triton X-100, the enzyme could be stored at -80 degrees C for several months.     

依赖雄激素的大鼠储精囊分泌蛋白的离子交换层析纯化

本文报道利用阳离子交换层析,纯化了雄激素依赖的大鼠储精囊分泌蛋白(SVPⅡ、SVPⅣ、SVPⅤ_a、SVPⅤ_b及SVPⅥ)。主要纯化步骤包括下列二步:1.Sep-hadex G-100凝胶过滤;2.上样后,联合使用盐梯度和pH梯度,洗脱快速蛋白液相层析(FPLC)系统的阳离子交换柱Mono S。洗脱峰的纯度以变性条件下的聚丙烯酰胶凝胶电泳(SDS-PAGE)和等电聚焦(IEF)鉴定;借此,还测定了已纯化的大鼠储精囊分泌蛋白的分子量和等电点。     

A ribosomal protein S6 kinase copurifies with phosphatase-1 activating factor

A highly purified preparation of phosphatase-activating kinase (Fa) from rabbit skeletal muscle phosphorylated ribosomal protein S6. The two activities copurified on DEAE-Sephadex, CM-Sephadex, and phosphocellulose chromatography and upon further chromatography on Sephacryl S-300 and FPLC Mono-S and Mono-Q columns. On the latter column, two separate peaks of Fa activity were observed when it was developed in Tris buffer as opposed to beta-glycerophosphate. S6 kinase activity was obtained only with the Fa which adhered to the resin. The Mr of the Fa and S6 activities was determined to be 83,200 by gel permeation on a Sephacryl S-300 column. The Fa preparation phosphorylated serine residues on S6; two tryptic phosphopeptides, A and C, were identified by two-dimensional phosphopeptide analysis. The enzyme also showed good activity toward initiation factor eIF-4B. Based on specificity toward ribosomal proteins and initiation factors, the Fa and a mitogen-stimulated S6 kinase purified from insulin-stimulated 3T3-L1 cells were similar. These results suggest that a form of Fa and an insulin-stimulated S6 kinase may be related or closely associated.     

Estrogen sulfotransferase of mouse placenta: behaviour and characteristics during partial purification

Mouse placental estrogen sulfotransferase (ST) was partially purified by fast protein liquid chromatography (FPLC) gel filtration in combination with FPLC anion exchange. Owing to the highly unstable nature of the enzyme, large increases in specific activity were not obtained. Storage of the ST in the presence of thiol groups at -20 degrees C stabilized the enzyme considerably. Forty-three percent of the cytosolic ST was bound to an Affi-Gel blue column and eluted as a broad peak at approximately 0.8 M NaCl. The use of the latter procedure, in combination with FPLC gel filtration, did not increase the specific activity substantially. Larger increases in specific activity were obtained using agarose-hexane-adenosine-3',5'-diphosphate affinity chromatography. The bound ST activity was eluted under a single peak at 1 mM ADP. Increases in specific activity following use of this column averaged 54-fold but could reach 90-fold. Attempts at further purification of this material resulted in low recovery and decreased specific activity. Velocity versus substrate concentration curves show that estrone and particularly estradiol inhibit the partially purified mouse placental sulfotransferase above 0.1-0.25 microM substrate concentrations.     

Rapid separation of Escherichia coli 30S ribosomal proteins by fast protein liquid chromatography (FPLC)

The proteins of the 30S ribosomal subunit from Escherichia coli have been separated by reverse-phase high-performance liquid chromatography on a short alkyl chain (C1/C8)-coated phase. The reverse-phase column was connected to a fast protein liquid chromatography (FPLC) system. The 21 proteins of the 30S ribosomal subunit were resolved into 16 peaks. Eleven proteins were isolated in purified form in a single chromatographic run as shown by polyacrylamide gel electrophoresis and amino acid analysis. Interestingly, the retention times of some proteins differed from the retention times observed on other reversed-phase support materials. The results show the speed and resolution of reverse-phase FPLC for both analytical and semi-preparative separations of 30S ribosomal proteins.     

Purification and biochemical characterization of antioxidant peptide from horse mackerel (Magalaspis cordyla) viscera protein

In the present study, a peptide having high antioxidant properties was isolated from horse mackerel viscera protein, Magalaspis cordyla. In vitro gastrointestinal digestion was employed to obtain potential protein hydrolysate and was subjected to consecutive chromatographic methods using fast protein liquid chromatography (FPLC) connected to diethyl amino ethyl (DEAE) anion exchange column and Sephadex G-25 gel filtration column. The activity of the fractions was tested against DPPH and hydroxyl radicals and the isolated peptide showed 89.2 and 59.1 percentage of scavenging. The amino acid sequence of purified peptide was determined using ESI-MS/MS as Ala-Cys-Phe-Leu (518.5 Da), it exhibited high activity against polyunsaturated fatty acid (PUFA) peroxidation than that of natural antioxidant, α-tocopherol.     

Purification and characterization of protein synthesis initiation factors IF1, IF2, and IF3 from Escherichia coli.

A simple procedure is described for the purification in high yields of protein synthesis initiation factors IF1, IF2, and IF3 from Escherichia coli strain MRE 600. IF2 was separated from IF1 and IF3 by ammonium sulfate fractionation and was purified by column chromatography on phosphocellulose and diethylaminoethyl (DEAE) Sephadex. IF1 and IF3 were separated by phosphocellulose column chromatography. IF1 was purified by molecular sieve chromatography, and IF3 by phosphocellulose column chromatography in urea buffer. Each factor was analyzed by sodium dodecyl sulfate or urea polyacrylamide gel electrophoresis and was greater than 98% pure. Only one form of IF1 and IF3 was found, with molecular weights of 8,500 and 22,500, respectively. Two forms of IF2 were isolated: IF2a with a molecular weight of 118,000 and IF2b with a molecular weight of 90,000. The amino acid composition of each factor was determined, and their stimulation in a variety of assays for initiation of protein synthesis is reported.     

An automatic multidimensional chromatography system for purification of human uterine progesterone receptor and induction of polyclonal antibodies

This paper reports on the synthesis of Org2058-bonded microparticulate silicas and their use in affinity chromatography as the first step for the purification of human progesterone receptor. The development of microprocessor-controlled instruments allows all the various steps to be performed automatically. The various steps used for the purification of human progesterone receptor were carried out with the FPLC system: affinity chromatography, desalting of eluate on Sephadex G-25, anion-exchange chromatography using a Mono Q column. With this procedure the receptor was purified approx. 10,000-fold within 24 h. The yield of receptor was generally 85-95%. Investigations with induced anti-progesterone receptor antibodies obtained after the fourth immunization show their immunoreactive behaviour towards progesterone receptor in crude cytosol, which was proved by sucrose density gradient centrifugation and by gel filtration on the FPLC system using a Sepharose 12 column. This implies that progesterone receptor was efficiently purified by our purification procedure.     

Phospholipid transfer protein from maize seedlings is partly membrane-bound

Specific antibodies raised against phospholipid transfer protein from maize seeds, react with mitochondria or microsomes solubilized by sodium deoxycholate. A single precipitin line was observed with both types of solubilized membranes when the double immunodiffusion technique was used. When the solubilized membranes were separated by fast-protein liquid chromatography (FPLC) on a reverse phase column, prior to the immunodiffusion, only fractions co-migrating with the pure phospholipid transfer protein, reacted with the antibody. Alternatively, solubilized membranes were submitted to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by immunoblotting; the detection of antigen-antibody complexes by a peroxydase reagent revealed the presence of a band co-migrating with the pure protein. All these observations strongly suggest that phospholipid transfer proteins are membrane-bound. The physiological significance of this finding will be discussed.     

Extracellular calmodulin-binding proteins in plants: purification of a 21-kDa calmodulin-binding protein

A 21-kDa calmodulin (CaM)-binding protein and a 19-kDa calmodulin-binding protein were detected in 0.1 M CaCl₂ extracts of Angelica dahurica L. suspension-cultured cells and carrot (Daucus carota L.) suspension-cultured cells, respectively, using a biotinylated cauliflower CaM gel-overlay technique in the presence of 1 mM Ca²⁺. No bands, or very weak bands, were shown on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels overlayed with biotinylated cauliflower CaM when 1 mM Ca²⁺ was replaced by 5 mM EGTA, indicating that the binding of these two CaM-binding proteins to CaM was dependent on Ca²⁺. Less 21-kDa CaM-binding protein was found in culture medium of Angelica dahurica suspension cells; however, a 21-kDa protein was abundant in the cell wall. We believe that the 21-kDa CaM-binding protein is mainly in the cell wall of Angelica dahurica. Based on its reaction with periodic acid-Schiff (PAS) reagent, this 21-kDa protein would appear to be a glycoprotein. The 21-kDa CaM-binding protein was purified by a procedure including Sephadex G-100 gel filtration and CM-Sepharose cation-exchange column chromatography. The purity reached 91% according to gel scanning. The purified 21-kDa CaM-binding protein inhibited the activity of CaM-dependent NAD kinase and the degree of inhibition increased with augmentation of the 21-kDa protein, which appeared to be the typical characteristic of CaM-binding protein.     

Isoelectric precipitation and gel chromatography for purification of monoclonal IgM

A preparative scale purification procedure of monoclonal IgM from hybridoma culture supernatant with high protein content is described. The procedure consists of three steps starting with ultrafiltration followed by isoelectric precipitation and gel chromatography. Cells and debris from culture supernatant were removed by microfiltration. The clarified supernatant was concentrated 400-fold in a hollow fibre ultrafiltration apparatus (cut off 100 000 daltons). The concentrate was titrated with dilute histidine/HCl buffer close to the isoelectric point of the IgM. Precipitated proteins were harvested by centrifugation, washed and redissolved. The protein fraction containing the IgM was further purified by gel chromatography on a Sephacryl S300 column. This procedure leads to product recovery of 40% and purity of 99% related to total protein.     

Simultaneous isolation of protein C activator,fibrin clot promoting enzyme (fiprozyme) and phospholipase A2 from the venom of the southern copperhead snake

The simultaneous isolation of three enzymes from the southern copperhead snake venom (Agkistrodon contortrix contortrix; ACC) is described. The first step is a chromatography of crude venom on a Mono S cation-exchange column at pH 6.5. A fibrin clot promoting enzyme (fiprozyme) that preferentially releases fibrinopeptide B from fibrinogen is isolated from the fraction not binding to the Mono S by a further three-step process. The procedure involves affinity chromatography on Blue Sepharose, gel chromatography on Sephacryl S-200 and metal–chelate chromatography on Chelating Sepharose. Protein C activator and phospholipase coelute from the Mono S column. They are separated by a gel chromatography on Sephacryl S-200. After this step two enzymes are obtained: a highly purified protein C activator applicable in methods for determination of functional level of protein C (a plasma regulator of hemostasis) and an electrophoretically pure enzyme with the activity of phospholipase A₂.     

The purification of cytochrome f and plastocyanin using affinity chromatography

Both plastocyanin and cytochrome f were purified using a combination of affinity chromatography together with established methods. Plastocyanin was partially purified using the method of Davis and San Pietro (Anal. Biochem. 95 (1979) 254-259), after which it was further purified using a column of cytochrome c covalently attached to Sepharose 4B. The affinity column was prepared using the method of Godinot and Gautheron (Methods Enzymol. 54 (1979) 112-114). The final purity index ratio (A278/A597) was less than 1.2, which is equal to that obtained using the more expensive FPLC procedure (Anderson, G.P., Sanderson, D.G., Lee, C.H., Durell, S., Anderson, L.B. and Gross, E.L. (1987) Biochim. Biophys. Acta 894, issue 3). Cytochrome f was partially purified using a modification of the method of Matazaki et al. (Plant Cell. Physiol. 16 (1975) 237-246) and bound to an affinity column of plastocyanin covalently attached to Sepharose 4B. Cytochrome f purified using this procedure had a purity index ratio (A554.5/A277) of 1.2. Both proteins are tyrosine proteins containing no tryptophan residues. After the affinity chromatography step, the fluorescence emission spectrum of either plastocyanin or cytochrome f was typical of a tyrosine protein free from tryptophan contamination.     

Purification and properties of chicken egg-white cobalamin-binding protein

A cobalamin-binding protein has been purified from chicken egg-white by using a combination of conventional and high performance ion-exchange chromatography. Following initial purification by DEAE-cellulose, ammonium sulphate precipitation, Sephacryl S-200 CM-cellulose and affinity chromatography, appropriate fractions were further purified using the Pharmacia fast protein liquid chromatography (FPLC) system. Using this method of purification, egg-white CBP has been purified more rapidly and with greater recovery than with conventional column chromatography. The homogeneity of this protein was verified by SDS-PAGE. The Mr was 37,000 by SDS-PAGE and 39,000 by gel filtration, which indicated that it was a glycoprotein. The stokes radius was 4.1 nm and pI was 4.3. The protein bound 57COB12 with a molar ratio of 1/1 and kd of 0.40 microM. The egg-white CBP was composed of 294 amino acid residues. Thiol groups and metal ions were not connected with the Cbl-binding activities.     

Purification and characterization of a cytosolic cytochrome P450 from the yeast Trichosporon cutaneum

A soluble cytochrome P450 from the yeast Trichosporon cutaneum was purified to homogeneity, using ammonium sulfate fractionation followed by fast protein liquid chromatography (FPLC) with DEAE-cellulose and phenyl-Sepharose columns. This procedure resulted in a 45-fold increase in specific activity with an activity yield of 6.8%. One- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the purified enzyme was homogeneous and had a molecular mass of 45 kDa. The purified enzyme contained a heme group and had a characteristic absorption peak at 448 nm in the reduced carbon monoxide difference spectrum. This enzyme was a monomeric protein and catalyzed the conversion of salicylic acid to catechol in the presence of NADH or NADPH. The N-terminal amino acid sequence indicated that the Trichosporon cutaneum cytochrome P450 did not show homology to most eukaryotic cytochromes P450, but had a high degree of homology to one cytochrome P450, the nitric oxide reductase, of Fusarium oxysporum.     

Nucleoside diphosphate kinase from haloalkaliphilic archaeon Natronobacterium magadii: purification and characterization

An ATP-binding protein from the haloalkaliphilic archaeon Natronobacterium magadii was purified and characterized by affinity chromatography on ATP-agarose and by fast protein liquid chromatography (FPLC) on a Mono Q column. The N-terminal 20 amino acid sequence of the kinase showed a strong sequence similarity of this protein with nucleoside diphosphate (NDP) kinases from different organisms and, accordingly, we believe that this protein is a nucleoside diphosphate kinase, an enzyme whose main function is to exchange γ-phosphates between nucleoside triphosphates and diphosphates. Comparison of the molecular weights of the NDP kinase monomer determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (23 000) and of the oligomer determined by sedimentation equilibrium experiments (125 000) indicated that the oligomer is a hexamer. The enzyme was autophosphorylated in the presence of [γ-³²P]ATP, and Mg²⁺ was required for the incorporation of phosphate. The kinase preserved the ability to transfer γ-phosphate from ATP to GDP in the range of NaCl concentration from 90 mM to 3.5 M and in the range of pH from 5 to 12. It was found and confirmed by Western blotting that this kinase is one of the proteins that bind specifically to natronobacterial flagellins. NDP kinase from haloalkaliphiles appeared to be simple to purify and to be a suitable enzyme for studies of structure and stability compared with NDP kinases from mesophilic organisms. Received: December 3, 1997 / Accepted: January 29, 1998     

First isolation of an antifungal lipid transfer peptide from seeds of a Brassica species

An antifungal peptide with a molecular mass of 9412 and an N-terminal sequence exhibiting notable homology to those of lipid transfer proteins was isolated from seeds of the vegetable Brassica campestris. The purification protocol entailed ion exchange chromatography on Q-Sepharose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography by fast protein liquid chromatography (FPLC) on Mono S, and gel filtration by FPLC on a Superdex peptide column. The antifungal peptide was adsorbed on Affi-gel blue gel and Mono S. It inhibited mycelial growth in Fusarium oxysporum and Mycosphaerella arachidicola with an IC(50) value of 8.3 microM and 4.5 microM, respectively. It exhibited dose-dependent binding to lyso-alpha-lauroyl phosphatidylcholine. The present findings constitute the first report on a non-specific lipid transfer protein from the seeds of a Brassica species.     

Ca2+-dependent protein kinase from alfalfa (Medicago varia): Partial purification and autophosphorylation

A calcium-dependent protein kinase (CDPK) was purified to 1400-fold from the soluble fraction of alfalfa (Medicago varia) cells by ammonium sulfate fractionation, Sephacryl-300, DEAE-Sephacel, Phenyl-Sepharose and Hydroxylapatite column chromatography. The enzyme is mainly monomeric. During the course of the purification steps a 50 kDa phosphoprotein doublet and a 56 kDa phosphoprotein copurified with the CDPK activity. Mobility shift of these proteins have been shown by SDS PAGE in Ca²⁺ free conditions. Tests on enzyme activity after separation by native gel electrophoresis revealed two protein kinase activities in our enzyme preparation and the phosphorylation of the 50 kDa and 56 kDa proteins. We suggest that these proteins are the autophosphorylated forms of calcium dependent protein kinases. Preincubation of the CDPK in ATP resulted in a marked increase in enzyme activity, but did not alter the Ca²⁺ sensitivity of the protein kinase.     

Simultaneous purification of DNA topoisomerase I and II from eukaryotic cells

We have developed a procedure for the simultaneous purification of DNA topoisomerase I and II from calf thymus. Both enzymes were first extracted from isolated nucleoprotein complexes. After batchwise chromatography on hydroxylapatite the two enzyme activities were separated on a FPLC phenylsuperose column. The enzymes were further purified by a second chromatography on phenylsepharose (topo I) or FPLC Mono Q (topo II). The purification can be finished within three days, yielding 0.5-1.0 mg quantities of homogeneous, enzymatic active preparations of the two proteins from 200 g of starting material.     

Production of native creatine kinase B in insect cells using a baculovirus expression vector

A full-length human creatine kinase B (B-CK) cDNA was used to produce a recombinant baculovirus (AcDZ1-BCK). Sf9 cells infected with this recombinant expressed a homodimeric protein composed of 43 kDa subunits which, under optimal conditions, formed up to 30% of the total soluble cellular protein. Upon analysis by PAGE, zymogram assay and gel filtration chromatography the recombinant protein behaved like authentic dimeric human BB-CK protein. Studies with a newly produced monoclonal antibody (CK-BYK/21E10) directed against an epitope in the N-terminus of the protein confirmed the identity of the product. The recombinant BB-CK protein was purified to over 99% homogeneity from the total protein extract of AcDZ1-CKB infected cells in one single step involving anion exchange column chromatography on MonoQ in FPLC. Dialysed protein had a specific activity of 239 U/mg protein.