Identifying a recombinant alkyldihydroxyacetonephosphate synthase suited for crystallographic studies

Alkyldihydroxyacetonephosphate is the building block for the biosynthesis of ether phospholipids, which are essential components of eukaryotic cell membranes and are involved in a variety of signaling processes. The metabolite is synthesized by alkyldihydroxyacetonephosphate synthase (ADPS), a peroxisomal flavoenzyme. Deficiency in ADPS activity causes rhizomelic chondrodysplasia punctata type 3, a very severe genetic disease. ADPS is unusual in that it uses a typical redox cofactor such as FAD to catalyze a non-redox reaction. With the goal of undertaking a structural investigation of the enzyme, we have characterized recombinant ADPS from different sources: Cavia porcellus, Drosophila melanogaster, Homo sapiens, Archaeoglobus fulgidus, and Dictyostelium discoideum. The protein from D. discoideum was found to be the best candidate for structural studies. We describe a protocol for expression and purification of large amounts of pure and stable enzyme in its holo (FAD-bound) form. A search of deletion mutants identified a protein variant that forms crystals diffracting up to 2A resolution.     

A Genetically Distinct Form of Cyclic Amp Phosphodiesterase Associated with Chromomere 3d4 in DROSOPHILA MELANOGASTER

Two cyclic AMP phosphodiesterase enzymes (E.C.3.1.4.17) are present in homogenates of adult Drosophila melanogaster. The two enzymes differ from one another in heat stability, affinity for Mg++, Ca++ activation and molecular weight. They do not differ markedly in their affinities for cyclic AMP, and both exhibit anomalous Michaelis-Menten kinetics. The more heat-labile enzyme is controlled in a dosage-dependent manner by chromomere 3D4 of the X chromosome and is absent in flies that are deficient for chromomere 3D4. Chromomere 3D4 is also necessary for the maintenance of normal cAMP levels, for male fertility, and for normal female fertility and oogenesis. The structural gene(s) for the more heat-stable enzyme is located outside of chromomeres 3C12-3D4. Whether 3D4 contains a structural gene, or a regulatory gene necessary for the presence of the labile enzyme, remains to be determined.     

Studies in metachromatic leukodystrophy: XV. Purification of normal and mutant arylsulfatase A from human liver

In this report we describe a method to purify both normal and abnormal (inactive) arylsulfatase A. The abnormal enzyme protein was isolated both from cases of late infantile and early juvenile forms of metachromatic leukodystrophy. Conventional protein isolation methods reported earlier were followed by size exclusion high-performance liquid chromatography in the final purification stages. Both the mutant enzyme and the normal enzyme had the same HPLC elution behavior. They thus appeared to self-associate in a similar pH-dependent fashion. Both could be followed by their reaction to a rabbit antibody to normal human arylsulfatase A. The amount of homogenous protein obtained from about 500 grams of liver was 300-400 micrograms.     

Juvenile hormone diol kinase. II. Sequencing, cloning, and molecular modeling of juvenile hormone-selective diol kinase from Manduca sexta

The gene sequence of Manduca sexta juvenile hormone diol kinase (JHDK) codes for an enzyme that has 59% sequence identity to Drosophila melanogaster sarcoplasmic calcium-binding protein-2 (dSCP2). JHDK and dSCP2 are similar to G-proteins with three conserved sequence elements involved in purine nucleotide binding. Both proteins contain two pairs of EF-hand motifs. Characterization and partial purification of the D. melanogaster homolog of M. sexta JHDK from adult D. melanogaster gave material with JHDK activity. This activity has an experimental pI and molecular mass that are nearly identical to those of dSCP2. Moreover, D. melanogaster phosphotransferase activity has very similar chromatographic retention in three systems compared with M. sexta JHDK. Substrate docking to three-dimensional models of JHDK has shown that the three conserved nucleotide-binding elements surround the putative substrate-binding site and align with conserved sequence elements of p21(Ras) and adenylate kinase. D. melanogaster dSCP2 is a homolog of M. sexta JHDK, and these proteins constitute a novel kinase family that binds nucleotides using the scaffold of an SCP (Protein Data Bank code ).     

Synthesis of Drosophila melanogaster alcohol dehydrogenase in yeast

Expression systems for the heterologous expression of Drosophila melanogaster alcohol dehydrogenase (ADH) in Saccharomyces cerevisiae have been designed, analyzed and compared. Four different yeast/Escherichia coli shuttle vectors were constructed and used to transform four different yeast strains. Expression was detectable in ADH- yeast strains, from either a constitutive promoter, yeast ADH1 promoter (ADCp), or a regulated promoter, yeast GALp. The highest amount of D. melanogaster ADH was obtained from a multicopy plasmid with the D. melanogaster Adh gene expressed constitutively under the control of yeast ADCp promoter. The D. melanogaster enzyme was produced in cell extracts, as assessed by Coomassie blue staining and Western blotting after polyacrylamide-gel electrophoresis and it was fully active and able to complement the yeast ADH deficiency. Results show that D. melanogaster ADH subunits synthesized in yeast are able to assemble into functional dimeric forms. The synthesized D. melanogaster ADH represents up to 3.5% of the total extracted yeast protein.     

Expression of eukaryotic glycosyltransferases in the yeast Pichia pastoris

The methylotrophic yeast Pichia pastoris is often used as an organism for the heterologous expression of proteins and has been used already for production of a number of glycosyltransferases involved in the biosynthesis of N- and O-linked oligosaccharides. In our recent studies, we have examined the expression in P. pastoris of Arabidopsis thaliana and Drosophila melanogaster core alpha1,3-fucosyltransferases (EC 2.4.1.214), A. thaliana beta1,2-xylosyltransferase (EC 2.4.2.38), bovine beta1,4-galactosyltransferase I (EC 2.4.1.38), D. melanogaster peptide O-xylosyltransferase (EC 2.4.2.26), D. melanogaster and Caenorhabditis elegans beta1,4-galactosyltransferase VII (SQV-3; EC 2.4.1.133) and tomato Lewis-type alpha1,4-fucosyltransferase (EC 2.4.1.65). Temperature, cell density and medium formulation have varying effects on the amount of activity resulting from expression under the control of either the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) or inducible alcohol oxidase (AOX1) promoters. In the case of the A. thaliana xylosyltransferase these effects were most pronounced, since constitutive expression at 16 degrees C resulted in 30-times more activity than inducible expression at 30 degrees C. Also, the exact nature of the constructs had an effect; whereas soluble forms of the A. thaliana xylosyltransferase and fucosyltransferase were active with N-terminal pentahistidine tags (in the former case facilitating purification of the recombinant protein to homogeneity), a C-terminally tagged form of the A. thaliana fucosyltransferase was inactive. In the case of D. melanogaster beta1,4-galactosyltransferase VII, expression with a yeast secretion signal yielded no detectable activity; however, when a full-length form of the enzyme was introduced into P. pastoris, an active secreted form of the protein was produced.     

Magnetic DNA affinity purification of ecdysteroid receptor.

A new method for rapid purification to near homogeneity of the ecdysteroid receptor (EcdR) from Drosophila melanogaster nuclear extract is presented. In the first step of the purification procedure the EcdR molecules were radiolabelled with [3H]ponasterone A and the [3H]ponasterone A-EcdR complexes were chromatographed under very mild conditions on Fractogel EMD TMAE(s) ion-exchanger. A 23-fold purified receptor was obtained which can be stored in liquid N2 without loss of activity. The second step involved the use of a magnetic DNA affinity technique where the double stranded hsp 27 oligonucleotide containing EcdR binding sequence was biotin 5'-end labelled and bound to monodisperse superparamagnetic particles coated with streptavidin (Dynabeads M-280 Streptavidin) giving magnetic DNA affinity beads. The chromatographed EcdR-ponasterone A complexes were bound to the magnetic DNA affinity beads and by magnetic separation, wash and elution, a 29,000-fold enriched EcdR preparation was obtained within 1.5 h. This procedure can be applied for other EcdR sources with minor modifications.     

Single-step purification of superoxide dismutase from bovine erythrocytes by immunosorbent column

A simple and rapid procedure for the purification of superoxide dismutase (SOD) from bovine erythrocytes by means of affinity-purified anti-SOD antibodies coupled to Sepharose 4B is reported. The method allowed 80% recovery of electrophoretically pure enzyme directly from hemolyzed red blood cells. The mild elution conditions (3.5 M MgCl2) and the stability of the immunoadsorbent permit the column to be reused several times (greater than 50). The physicochemical properties of the enzyme obtained by this method are identical to those of the native protein.     

A method of isolation of partially purified alkaline peptide hydrolase from Drosophila melanogaster larvae

A semipreparative method is developed for preparing peptidohydrolase from Drosophila melanogaster larvae which involves the stages of extraction, salting-out, gel-filtration and ion-exchange chromatography. It is established that the maximal (up to 81%) yield of the enzyme is observed with the single extraction in the alkaline medium. The main bulk of the enzyme is salted-out in the low acid 3 M ammonium sulphate solution. Gel-filtration on column with Sephadex-25 provides complete salting-out of the enzyme-containing fraction, and ion exchange chromatography on CM-cellulose--a considerable purification of the enzyme under study. A degree of the obtained purification of the enzyme under study. A degree of the obtained peptidohydrolase preparation purity in acid and alkaline medium is determined by the method of electrophoresis in PAAG. At all stages of the preparation the enzyme possesses the casein-lytic activity and is able of hydrolyzing the ethyl ester and benzoyl arginine p-nitroanilide.     

High-performance liquid chromatography of proteins on deformed nonporous agarose beads. Affinity chromatography of dehydrogenases based on cibacron blue-derivatized agarose

Nonporous agarose beads, prepared by shrinkage and cross-linking in organic solvents, were derivatized with Cibacron Blue F3G-A. A compressed bed of these beads was used for purification of dehydrogenases (glucose-6-phosphate dehydrogenase, lactate dehydrogenase and alcohol dehydrogenase). The chromatographic conditions for the purification of glucose-6-phosphate dehydrogenase were optimized by varying the pH of the buffer; the concentrations of eluting agents, i.e. NADP (specific elution) and sodium chloride (nonspecific elution); flow rate; residence time of the protein on the column bed; and protein load. Specific elution with NADP (2 mM in 0.025 M Tris-HCl, pH 8.0) gave the highest recovery (140%) and highest purification factor (200-fold) of the enzyme. The ability of the compressed bed of nonporous agarose beads to tolerate high flow rates was essential, since the recovery of the enzyme activity increased with an increase in flow rate.     

金属螯合亲和层析介质用于六聚组氨酸融合蛋白的纯化研究

以Sepharose CL-6B为载体,环氧氯丙烷为活化剂,羧甲基天冬氨酸(CM-Asp)为螯合配基制备载有Co2 的金属螯合亲和层析介质Co-CM-Asp-Sepharose,并将其用于六聚组氨酸融合蛋白的纯化研究。对纯化200μL细胞裂解液中靶蛋白所需Co-CM-Asp-Sepharose介质用量,Co-CM-Asp-Sepharose与细胞裂解液的孵育时间,介质清洗条件及靶蛋白洗脱时所需咪唑浓度等进行了优化。比较了Co-CM-Asp-Sepharose与Ni-NTA-Agarose(Qiagen公司)两种螯合介质对融合蛋白的纯化效果,开展了从5mL细胞裂解液中放大规模纯化融合蛋白的研究,并通过Bradford法测定了Co-CM-Asp-Sepharose对CD155D1蛋白的纯化量。结果表明:对200μL细胞裂解液纯化体系,Co-CM-Asp-Sepharose(50%悬浮液)的优选体积为60μL,最佳孵育时间为30min,洗脱液最佳咪唑浓度为200mmol/L,纯化得到融合蛋白的量约为200μg。介质用量放大为1.5mL(50%悬浮液)对CD155D1蛋白的纯化量可达4.6mg。与商品化Ni-NTA-Agarose相比,本介质具有选择性好,清洗条件简单,得到的靶蛋白纯度高等优点。     

High-Performance Liquid Chromatography of Proteins on Deformed Nonporous Agarose Beads. Affinity Chromatography of Dehydrogenases Based on Cibacron Blue-Derivatized Agarose

Nonporous agarose beads, prepared by shrinkage and cross-linking in organic solvents, were derivatized with Cibacron Blue F3G-A. A compressed bed of these beads was used for purification of dehydrogenases (glucose-6-phosphate dehydrogenase, lactate dehydrogenase and alcohol dehydrogenase). The chromatographic conditions for the purification of glucose-6-phosphate dehydrogenase were optimized by varying the pH of the buffer; the concentrations of eluting agents, i.e. NADP (specific elution) and sodium chloride (nonspecific elution); flow rate; residence time of the protein on the column bed; and protein load. Specific elution with NADP (2 mM in 0.025 M Tris-HCl, pH 8.0) gave the highest recovery (140%) and highest purification factor (200-fold) of the enzyme. The ability of the compressed bed of nonporous agarose beads to tolerate high flow rates was essential, since the recovery of the enzyme activity increased with an increase in flow rate.     

Purification of human adult and foetal intestinal alkaline phosphatases by monoclonal antibody immunoaffinity chromatography.

We have used the technique of monoclonal antibody immunoaffinity chromatography to purify adult and foetal intestinal alkaline phosphatases. Pure adult intestinal enzyme was obtained from a crude tissue extract with a single immunoaffinity chromatographic step in yields exceeding 95%. An additional ion-exchange chromatographic step was necessary for purification of the foetal enzyme, but yields still exceeded 70%. Experiments to optimize the efficiency of the monoclonal antibody immunoaffinity chromatography procedure suggest that the relative strength of binding of an antibody to its antigen is the most important factor to consider when constructing such columns. A column made from an antibody of too low an avidity will not retain the enzyme, while one of too high an avidity will make elution of enzyme in the active state difficult. A scheme is suggested for the application of this technique to a general approach to enzyme purification.     

Efficiency of a smooth desorption procedure for the purification of barley beta-amylase using immunoaffinity chromatography

Immunoaffinity chromatography was used for a one step purification procedure of beta-amylase from the G25 Sephadex gel filtrated fraction of whole barley protein extracts. The immunoglobulin G (IgG) fraction of an anti-barley beta-amylase immune serum was immobilized on Ultrogel. A gentle desorption procedure was used, combining distilled water elution with an interrupted elution. The quality of the purification was assayed by using cross immunoelectrophoresis with a polyspecific anti-barley protein immune serum. The extent of the damaging effect of this procedure was evaluated on the specific activity of the enzyme and on its polymorphism, as displayed by isoelectric focusing. The results underline the efficiency of the purification procedure and its low denaturing effect on the beta-amylase. This opens new possibilities for some aspects of the enzyme study and for the purification of other biologically active proteins.     

A comprehensive study of genic variation in natural populations of Drosophila melanogaster. VII. Varying rates of genic divergence as revealed by two-dimensional electrophoresis.

Four sibling species from the melanogaster subgroup (Drosophila melanogaster, D. simulans, D. sechellia, and D. mauritiana) were studied for genetic divergence, by high-resolution two-dimensional protein electrophoresis (2DE) coupled with ultrasensitive silver staining. A total of eight tissues from larval and adult developmental stages representing both gonadal (germ-line) and nongonadal (somatic) tissues were analyzed for protein divergence between species. Close to 400 polypeptides (protein spots) were scored from each tissue and species, and protein divergence was measured on the basis of qualitative differences (presence/absence) of protein spots in pairwise species comparisons. The observed levels of genic divergence varied among tissues and among species. When larval hemolymph proteins (which are known to be highly polymorphic) were excluded, there was no evidence to suggest that either the larval or adult-stage proteins, as a whole, are more diverged than the other; variation between different tissues rather than between developmental stages appears to be the most significant factor affecting genetic divergence between species. The reproductive tissue (testis and accessory gland) showed more divergence than did the nonreproductive tissue; D. melanogaster testis (from both larvae and adult males) showed the highest level of divergence. In view of the previous observation that D. simulans, D. mauritiana, and D. sechellia show similar but significantly less reproductive isolation from each other than from D. melanogaster, the present results suggest a correlation between the levels of reproductive-tract-protein divergence and the degree of reproductive isolation in these species.     

An improved method for the purification of DNA-dependent RNA polymerase from Escherichia coli

DNA-dependent RNA polymerase from Escherichia coli was purified further by elution through heparin-Sepharose CL-6B column after the enzyme was obtained, partially purified, using Burgess and Jendrisak's method [(1975)Biochemistry 14, 4634] The total yield of the pure protein was 10 mg from 50 g of E.coli cells. The method was found to be very reproducible and convenient. The enzyme preparation had 60% active molecules and the elongation rate of RNA synthesis by this enzyme was measured to be 11 bases/s over delta D111 T7 DNA.     

Isolation to homogeneity and partial characterization of a histo-blood group A defined Fuc alpha 1----2Gal alpha 1----3-N-acetylgalactosaminyltransferase from human lung tissue

The soluble histo-blood group A glycosyltransferase (Fuc alpha 1----Gal alpha 1----3-N-acetylgalactosaminyltransferase) was purified approximately 600,000-fold to homogeneity from human lung tissue. The enzyme was solubilized in 1% Triton X-100, partially purified by affinity chromatography on Sepharose 4B, and eluted with UDP. Final purification was obtained by twice repeated fast protein liquid chromatography ion exchange (Mono STM) with NaCl gradient elution and reverse-phase chromatography (proRPC) with acetonitrile gradient elution. Identity of the purified protein was established by (i) demonstration of the putative A transferase protein only in affinity-purified extracts of A but not O individuals, and (ii) specific immunoprecipitation of enzyme activity and putative protein with monoclonal antibodies. Sodium dodecyl sulfate electrophoresis revealed a single protein band with apparent Mr of approximately 40,000 under both reducing and nonreducing conditions. Digestion with N-glycanase yielded a reduction in Mr of approximately 6,000 (estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis), suggesting that the A transferase is a glycoprotein with N-linked carbohydrate chains. Amino acid composition and N-terminal amino acid sequence of the intact transferase, as well as of peptides released by endolysyl peptidase digest or cyanogen bromide cleavage, are presented.