Purification and characterisation of tyrosine decarboxylase and aromatic-L-amino-acid decarboxylase

Microbial tyrosine decarboxylase (EC 4.1.1.25) and mammalian aromatic-L-amino-acid decarboxylase (EC 4.1.1.28) catalyse the formation of tyramine from L-tyrosine. These enzymes were characterised after isolation to purity by methods including fast polymer liquid chromatography (FPLC). Tyrosine decarboxylase was isolated from Streptococcus faecalis by FPLC anion exchange chromatography (11-times purification; 72% recovery; 23.2 U/mg protein). FPLC on Phenyl-Superose resulted in purification to 115 U/mg protein. Aromatic-L-amino-acid decarboxylase was isolated from pig kidney by ammonium sulfate fractionation, DEAE chromatography, and FPLC anion exchange chromatography (21-times purification; 22% recovery; 0.71 U/mg protein). By FPLC chromatofocusing, tyrosine decarboxylase eluted at pH 4.3 and aromatic-L-amino-acid decarboxylase at pH 5.0. Isoelectric focusing of tyrosine decarboxylase gave two bands (pI 4.4 and 4.5). With pyridoxal 5'-phosphate removed by ultrafiltration, only one band (pI 4.4) appeared, and SDS polyacrylamide electrophoresis confirmed the purity. FPLC gel filtration resulted in molecular weights 143,000 and 86,000, respectively, for tyrosine decarboxylase and aromatic-L-amino-acid decarboxylase. In SDS electrophoresis, tyrosine decarboxylase had the monomer molecular weight 75,000, showing a dimer structure for the enzyme.     

Purification of plasmid DNA by fast protein liquid chromatography on superose 6 preparative grade

We were able to reduce both the time and the use of hazardous chemicals associated with the previous plasmid isolation methods of high-pressure liquid chromatography and CsCl gradient centrifugation by employing fast protein liquid chromatography (FPLC). Plasmid was first crudely prepared from bacterial cultures by a standard alkaline lysis method. After an alcohol precipitation, the nucleic acids were divided into two equal portions. One half was used for a standard purification method employing CsCl centrifugation. The other was dissolved in FPLC buffer, treated with RNase A, and applied to a Superose 6 preparative grade column (HR 10/30). Plasmid eluted off the column within 20 min as a single, highly resolved peak. Plasmid isolated by FPLC had yields, purity, and transformation efficiencies similar to that isolated by CsCl centrifugation.     

限制性内切酶EcoR的高效表达与纯化

EcoR Ⅱ was the first restriction endonuclease ever found requiring the cooperative interaction with the least two DNA sites for digestion activity.To study the specific activity, Eco R Ⅱ was purified from hyperexpression engineering bacteria in which the specific expression products increased to about 20% of total cellular protein.By using chromatography on DEAE cellulose column,phosphocellulose column and FPLC of Resource Q,the enzyme was purified from soluble protein fraction.The inclusion bodies were solved and renatured,and the enzyme was purified from this part of protein with higher specific activity by using FPLC of Resource Q.Detection showed that the enzyme was purified to homogeneity and was free of detectable contamination by other DNase(exo and endo).     

Carboxylmethylation of Calmodulin in Cultured Pituitary Cells

We have used fast protein liquid chromatography (FPLC) and reverse-phase HPLC to rapidly resolve carboxylmethylated proteins in cultured pituitary GH3 cells. This procedure preserves labile carboxylmethyl esters, which are lost under the usual procedures employed for protein fractionation. GH3 cells were incubated with [methyl-3H]-methionine in culture and incorporation of label into the soluble fraction, total cell protein, and protein carboxylmethyl esters was determined; protein carboxylmethyl ester formation was shown to be resistant to cycloheximide. Fractionation of protein carboxylmethyl esters from GH3 cells by gel permeation FPLC, anion-exchange FPLC, and reverse-phase HPLC in the presence of calcium and in the presence of EGTA identified two proteins that are major substrates for protein carboxylmethyltransferase and indicated that one of these proteins is calmodulin. Similar results were obtained when a cytosolic fraction from GH3 cells was incubated with S-adenosyl-L-[methyl-3H]methionine. These results indicate that rapid chromatography at low temperature and low pH is useful for the analysis of eucaryotic carboxylmethylated proteins and that contrary to reports obtained in other systems, calmodulin is carboxylmethylated in intact pituitary cells.     

Purification of ribulose-1, 5-bisphosphate carboxylase/oxygenase with high specific activity by fast protein liquid chromatography

A rapid procedure for the purification of ribulose-1, 5-bisphosphate carboxylase/oxygenase (rubisco) (EC 4.1.1.39) by fast protein liquid chromatography (FPLC) is described. Chloroplasts isolated mechanically from spinach leaves were used as the source of a stromal extract enriched in rubisco. By subsequent fractionation of this extract on ion-exchange FPLC, highly purified rubisco (sp act 2.10-2.76 mumol/mg protein X min) was obtained in less than 30 min. The high specific activity and excellent stability of the final preparation can be attributed to the use of chloroplasts as a starting material and the short time required for the chromatographic separation, both of which minimize proteolytic activity.     

Purification and substrate specificity of an endopeptidase from the human oral spirochete Treponema denticola ATCC 35405, active on furylacryloyl-Leu-Gly-Pro-Ala and bradykinin.

An endopeptidase was purified to homogeneity from the cell extracts of Treponema denticola ATCC 35405 (a human oral spirochete) by a procedure that comprised dialysis, anion exchange fast protein liquid chromatography (FPLC), hydroxylapatite FPLC, immobilized metal affinity FPLC, FPLC chromatofocusing, and two consecutive gel permeation FPLC steps. The enzyme is a 62-kDa protein with an isoelectric point of 6.5-7.0. Experiments with enzyme inhibitors suggest that this enzyme is a metallopeptidase and that its activity is not dependent on sulfhydryl or serine residues. The enzyme is active on furylacryloyl-Leu-Gly-Pro-Ala (FALGPA; pH optimum near 6.25), bradykinin (Bk), and several Bk-related peptides. In FALGPA, the cleavage site is the Leu-Gly bond. An imino acid is absolutely necessary in position P'2. The shortest hydrolyzed peptide was FALGPA, the hydrolysis of which is strongly and competitively inhibited by Bk (Ki = 5.0 microM). The pyrophosphate ion and phosphoramidon also inhibited the hydrolysis of FALGPA. The enzyme does not hydrolyze all typical synthetic collagenase substrates, Azocoll, Azocasein, or Type I and Type IV collagens, or any other proteins tested. In Bk-related peptides, the hydrolyzed bond was Phe5-Ser6. Since a Bk antagonist and a Bk-potentiating pentapeptide also were good substrates, it is possible that the enzyme hydrolyzes Bks and related peptides only because of the coincidental, specific amino acid sequence of those substrates. A proposal is made that since a substantial portion of the amino acid sequence of FALGPA is present in collagen (and additionally acknowledging that the furylacryloyl residue structurally resembles that of proline), the natural substrates of this enzyme may be small, soluble collagen fragments produced by other enzymes from periodontal connective tissue, and that such peptides are important for the nutrition and pathogenicity of T. denticola.     

Purification of prenylated proteins by affinity chromatography on cyclodextrin-modified agarose

Although protein prenylation is widely studied, there are few good methods for isolating prenylated proteins from their nonprenylated relatives. We report that crosslinked agarose (e.g., Sepharose) chromatography medium that has been chemically functionalized with β-cyclodextrin (β-CD) is extremely effective in affinity chromatography of prenylated proteins. In this study, a variety of proteins with C-terminal prenylation target (“CAAX box”) sequences were enzymatically prenylated in vitro with natural and nonnatural prenyl diphosphate substrates. The prenylated protein products could then be isolated from starting materials by gravity chromatography or fast protein liquid chromatography (FPLC) on a β-CD-Sepharose column. One particular prenylation reaction, farnesylation of an mCherry-CAAX fusion construct, was studied in detail. In this case, purified farnesylated product was unambiguously identified by electrospray mass spectrometry. In addition, when mCherry-CAAX was prenylated with a nonnatural, functional isoprenoid substrate, the functional group was maintained by chromatography on β-CD-Sepharose, such that the resulting protein could be selectively bound at its C terminus to complementary functionality on a solid substrate. Finally, β-CD-Sepharose FPLC was used to isolate prenylated mCherry-CAAX from crude HeLa cell lysate as a model for purifying prenylated proteins from cell extracts. We propose that this method could be generally useful to the community of researchers studying protein prenylation.     

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 of a high molecular weight membrane protein by fast protein liquid chromatography: the ATPase complex of a thermophilic cyanobacterium

Fast protein liquid chromatography (FPLC) with a strong anion-exchange (Mono Q) column is applied to the purification of a high molecular weight membrane protein. The ATPase complex of the thermophilic cyanobacterium Synechococcus 6716, partially purified by ammonium sulfate precipitation, was fractionated in the presence of the detergent octylglucoside. The ATPase complex containing fractions were eluted by a linear NaCl gradient at about 0.4 M and within 10 min. The FPLC fractions were analyzed for protein and pigment contents and by polypeptide composition. The purest fraction is essentially free of pigments and has a high specific ATP hydrolysis activity (about 1.6 mumol ATP X min-1 X mg protein-1) which is sensitive to N,N'-dicyclohexylcarbodiimide.     

Heterologous expression, purification, and properties of a potato protein inhibitor of serine proteinases

The gene PKPI-B10 [AF536175] encoding in potato (Solanum tuberosum L., cv. Istrinskii) a Kunitz-type protein inhibitor of proteinases (PKPI) has been cloned into the pET23a vector and then expressed in Escherichia coli. The recombinant protein PKPI-B10 obtained as inclusion bodies was denatured, separated from admixtures by ion-exchange fast protein liquid chromatography (FPLC) on MonoQ under denaturing conditions, and renatured. The native protein was additionally purified by ion-exchange FPLC on DEAE-Toyopearl. The PKPI-B10 protein effectively inhibits the activity of trypsin, significantly weaker suppresses the activity of chymotrypsin, and has no effect on other serine proteinases: human leukocyte elastase, subtilisin Carlsberg, and proteinase K, and also the plant cysteine proteinase papain.     

FPLC分离鉴定寡脱氧核苷酸片段

机器合成的反义核酸药物需要有效的鉴定纯度方法,用ＭＯＮＯ－Ｑ柱在ｐＨ１２时以ＮａＣｌ浓度度洗脱,可以将１９－２１个碱基的小片段寡核苷到很好地分开,因此快速蛋白质液相色谱（ＦＰＬＣ）可以用来分离鉴定反义核酸药物。     

Chitinase-like proteins with antifungal activity from emperor banana fruits

Two 30-kDa proteins with N-terminal sequence homology to chitinases have been isolated from fruits of the emperor banana by using a protocol that involved (NH(4))(2)SO(4) precipitation, 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 Superdex 75. The proteins were adsorbed on Affi-gel blue gel and Mono S. They both inhibited mycelial growth in Fusarium oxysporum but not in Mycosphaerella arachidicola. The chitinase-like protein more strongly bound on Mono S was obtained with a slightly lower yield and exhibited a higher antifungal potency toward F. oxysporum when compared with the less strongly bound chitinase-like protein.     

Valorization of corn cob for the obtention and purification of endoglucanase produced by SSF

Agro-food by-products contain valuable nutrients which are wasted. In this work, solid-state fermentation (SSF) was carried out using corn cob as substrate for endoglucanase production. The radial growth of three fungal strains -Trichoderma harzianum T104, Aspergillus niger GH1 and Aspergillus niger NRRL3- was analyzed in order to select the most appropriate. Radial growth data were analyzed with a mixed linear model for longitudinal data and no statistically significant differences were found between both A. niger strains.Endoglucanase was separated from the extract of A. niger GH1 by fast protein liquid chromatography (FPLC).The highest endoglucanase activity was detected in fraction number three collected from FPLC corresponding to 72 h of SSF. Seven bands in a range from 24 to 50 kDa, which correspond to endoglucanase from fungal extract, were detected by zymogram analysis. According to protein quantification performed by the ImageJ software, 85% of the proteins present in the samples collected by FPLC corresponded to endoglucanase proteins. The purified endoglucanase retained about 100% of its catalytic activity at 30 °C and 50 °C and was stable in a pH range between 4.00-6.00. These properties make this isolated enzyme suitable for industrial applications such as the saccharification process for bioethanol production.     

Separation of isoenzymes of citrate synthase and isocitrate dehydrogenase by fast protein liquid chromatography

Fast protein liquid chromatography (FPLC) has been shown to be a rapid and effective method of separating isoenzymes of citrate synthase and isocitrate dehydrogenase in extracts of Pseudomonas aeruginosa and Acinetobacter calcoaceticus. The advantages of FPLC over conventional methods of fractionation are discussed and it is suggested that this may be a valuable and more general technique for isoenzyme resolution.     

Method for rapid separation of 3,5,3'-triiodothyroacetic acid in human serum by fast protein liquid chromatography

The 3,5,3'-triiodothyroacetic acid (TRIAC) has been approved as a valuable agent in the management of hyperthyroidism secondary to inappropriate secretion of thyrotropin. We have developed a fast protein liquid chromatography (FPLC) method for separation and quantification of TRIAC. Serum samples charged with TRIAC were extracted with methanol/ammonium acetate, the supernatants were evaporated to dryness, reconstituted in NaOH and injected on a reversed phase column for chromatography. For separation an isocratic elution method (methanol water; 0.1% trifluoroacetic acid) was used. The area under the curve (ml%) was compared with those of the calibration curves. Recoveries were 70 +/- 10.8%. TRIAC was eluted in 2.33 ml. Conclusively, the present method shows that TRIAC can be measured by FPLC and may be applied to the measurement of TRIAC in pharmacological studies.     

Characterization of the chloramphenicol acetyltransferase variants encoded by the plasmids pSCS6 and pSCS7 from Staphylococcus aureus.

The two 4.6 kb chloramphenicol resistance (CmR) plasmids pSCS6 and pSCS7, previously identified in Staphylococcus aureus from subclinical bovine mastitis, both encoded an inducible chloramphenicol acetyltransferase (CAT, EC 2.3.1.28). The pSCS6- and pSCS7-encoded CAT variants were purified by ammonium sulphate precipitation, ion-exchange chromatography and fast protein liquid chromatography (FPLC). Both native enzymes showed Mr values of 70,000 on FPLC and were composed of three identical subunits, each of Mr approximately 23,000. The CAT variants from pSCS6 and pSCS7 differed in their net charges and in their isoelectric points. The isoelectric point of the CAT from pSCS6 was pH 5.7 and that of the CAT from pSCS7 pH 5.2. Both CAT variants exhibited highest enzyme activities at pH 8.0. The Km values for chloramphenicol and acetyl-CoA of the CAT from pSCS6 were 2.5 microM and 58.8 microM, respectively, while those of the CAT from pSCS7 were 2.7 microM and 55.5 microM. Both CAT variants were relatively thermostable. The CAT from pSCS6 was less sensitive to mercuric ions than the CAT from pSCS7.     

Purification of two muscle enzymes by chromatography on immobilized ferric ions

Two enzymes, glycogen phosphorylase and lactate dehydrogenase, were purified simultaneously in a single step. Ferric ions immobilized on a chelating gel were used as the adsorbent. Adsorption and desorption steps were accomplished by changes in buffer composition. The recoveries were better than 80% and the capacities were about 5 mg of protein per milliliter of adsorbent. The procedure worked well both on a small and on a preparative scale. The homogeneity of the purified enzymes was checked by FPLC.     

Fractionation and recovery of whey proteins by hydrophobic interaction chromatography

A method for the recovery and fractionation of whey proteins from a whey protein concentrate (80%, w/w) by hydrophobic interaction chromatography is proposed. Standard proteins and WPC 80 dissolved in phosphate buffer with ammonium sulfate 1 M were loaded in a HiPrep Octyl Sepharose FF column coupled to a fast protein liquid chromatography (FPLC) system and eluted by decreasing the ionic strength of the buffer using a salt gradient. The results showed that the most hydrophobic protein from whey is α-lactalbumin and the less hydrophobic is lactoferrin. It was possible to recover 45.2% of β-lactoglobulin using the HiPrep Octyl Sepharose FF column from the whey protein concentrate mixture with 99.6% purity on total protein basis.     

The differential apoA-I enrichment of prebeta1 and alphaHDL is detectable by gel filtration separation

The aim of the study was to assess the isolation of HDL by fast protein liquid chromatography (FPLC) to perform kinetics studies of apolipoprotein (apo)A-I-HDL labelled with a stable isotope. Comparison between FPLC and ultracentrifugation has been made. ApoA-I-HDL kinetics were studied by infusion of [5.5.5-(2)H(3)]leucine for 14 h in five subjects. Using FPLC, prebeta(1) HDL and alphaHDL (HDL(2) and HDL(3)) were separated from 200 microl of plasma samples. Total HDL was isolated by sequential ultracentrifugation (HDL-UC). The tracer-to-tracee ratio was higher in prebeta(1) HDL than in total HDL-UC. The higher leucine enrichment found in total HDL-UC compared to alphaHDL suggested the existence of a mixture of apoA-I-HDL sub-classes. From this difference in enrichments, the turnover rate of total HDL-UC, usually assumed to be alphaHDL, was probably overestimated in previous studies. To our knowledge, this study is the first report which provides a convenient tool to distinguish enrichments of apoA-I in prebeta(1) HDL and alphaHDL from total HDL previously used for kinetic measurements. This original and new method should help to understand the kinetics of HDL in humans and the reverse cholesterol transport dynamics.