Purification and characterization of human plasminogen activator inhibitor type I expressed in Saccharomyces cerevisiae

The rapidly acting inhibitor of plasminogen activators, PAI-1, was produced intracellularly in Saccharomyces cerevisiae by using the ADH2 promoter to drive the expression of the human PAI-1 cDNA. Approximately 8 mg of human PAI-1 was produced per liter of confluent yeast culture. A purification scheme which resulted in 20% recovery of isolated PAI-1 from the broken yeast cell homogenate was devised. Yeast-derived human PAI-1 differs from endothelial-type PAI-1 isolated from HT1080 fibrosarcoma cells in that the recombinant inhibitor does not contain carbohydrate side chains. Nevertheless, the activity and other functional attributes of yeast-derived PAI-1 are similar to those exhibited by HT1080 fibrosarcoma cell-derived PAI-1. Hence, this study demonstrates that expression of human PAI-1 in yeast is a viable strategy for the production of ample quantities of this key modulator of plasminogen activator-mediated proteolysis.     

Purification and characterization of recombinant endoglucanase of Trichoderma reesei expressed in Saccharomyces cerevisiae with higher glycosylation and stability

Cel5A (endoglucanase II) of Trichoderma reesei was expressed in Saccharomyces cerevisiae then purified. Two components (C1 and C2) of recombinant Cel5A with different glycosylation were obtained. Purified C1 had a larger molecular mass (57 kDa) than that of the native Cel5A produced by T. reesei (48 kDa) due to the different extents of asparagines-linked glycosylation. There was no significant difference in enzymatic activity between the C1 and the native Cel5A from T. reesei. C1 treated with Endoglycosidase H had a molecular mass of 54 kDa and retained about 88% of its original activity. Unpurified C2 was larger form of hyperglycosylation proteins. Its molecular mass was larger than 85 kDa till up to 200 kDa. It still retained activity regardless of its magnitude molecular mass. With increased glycosylation extent of the enzyme components (C2 >C1 >native Cel5A), the pH range of activity become wider, and thermal stability become higher.     

DNA polymerase III from Saccharomyces cerevisiae. I. Purification and characterization

Yeast cells from a wild type or protease-deficient strain were lysed in the absence or presence of protease inhibitors and the extracts analyzed by analytical high pressure liquid chromatography on diethylaminoethyl silica gel. Conditions that inhibited protease action caused elution of a novel DNA polymerase activity at a position in the gradient distinct from the elution positions of both DNA polymerase I and II. In large scale purifications, this DNA polymerase, called DNA polymerase III, copurified with a single-stranded DNA dependent 3'-5' exonuclease activity, exonuclease III, to near homogeneity. Glycerol gradient centrifugation partially dissociated the complex to yield two peaks of exonuclease III activity, one at 7.7 S together with the DNA polymerase, and one at 4.0 S without polymerase activity. Gel filtration indicated that the complex has a molecular mass greater than 400 kDa. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that the complex consists of several subunits: 140, 62, 55, and 53 kilodaltons, some of which may be proteolysis products. The exonuclease component of the complex can excise single nucleotide mismatches providing a base-paired primer-template which can be elongated by the DNA polymerase. Under replication conditions, the complex exhibits a measurable turnover rate of dTTP to dTMP and it contains no primase activity. The enzymatic activities of the 3'-5' exonuclease are consistent with a proofreading function during in vivo DNA replication. A second exonuclease activity, exonuclease IV, separated from the complex late in the purification scheme. It degrades both single-stranded and double-stranded DNA in the 5'----3' direction.     

Purification and biochemical characterization of recombinant hirudin produced by Saccharomyces cerevisiae

Recombinant hirudin was produced by the yeast Saccharomyces cerevisiae using the alpha-pheromone prepro sequence to direct its secretion into the culture medium. The secreted hirudin was isolated to greater than or equal to 95% purity as measured by 205-nm absorbance integration from a reverse-phase chromatogram. One major activity peak corresponding to the complete, correctly processed molecule and two minor activity peaks corresponding to C-terminally truncated forms were identified. The primary structure of the major peak, determined by N-terminal sequencing of tryptic peptides, was that predicted from the cDNA sequence, and the molecular mass analyzed by fast atom bombardment mass spectrometry (FAB-MS) was 6892.6 (calculated 6892.5). UV spectral analysis suggested that, in contrast to the natural molecule, recombinant hirudin produced by S. cerevisiae is not sulfated.     

Purification and characterization of human caseinomacropeptide produced by a recombinant Saccharomyces cerevisiae

Caseinomacropeptide (CMP) is a biologically active polypeptide derived from the C-terminal of milk kappa-casein. CMP is heterogeneous since it is modified differently by glycosylation and phosphorylation after translation. Recently, recombinant human CMP (hCMP) has been produced as a secretory product in yeast. The present study aimed at the purification and characterization of recombinant hCMP. By sequential molecular cut-off ultrafiltration and anion-exchange chromatography, the recombinant hCMP in the culture broth could be purified to an HPLC purity over 94%. The authenticity of the purified hCMP was confirmed by sequence analysis of N-terminal amino acids. The recombinant hCMP was estimated to be 7.0kDa by SDS-PAGE, and showed a lower glycosylation than the natural bovine CMP.     

Purification and characterization of hexahistidine-tagged cyclohexanone monooxygenase expressed in Saccharomyces cerevisiae and Escherichia coli

Cyclohexanone monooxygenase (CMO) is a soluble flavoenzyme originally isolated from Acinetobacter spp. which carries out Baeyer-Villiger reactions with cyclic ketone substrates. In the present study we cloned the Acinetobacter CMO gene and modified it for facile purification from heterologous expression systems by incorporation of a His(6)-tag at its C-terminus. A single purification step employing metal (Ni(2+))-affinity column chromatography provided essentially homogeneous enzyme in yields of 69-72%. The properties of the purified, recombinant enzymes (rCMO) were compared with that of native CMO (nCMO) isolated from Acinetobacter cultures grown in the presence of cyclohexanone. The specific activities of His(6)-tagged rCMO and nCMO toward their index substrate, cyclohexanone, were similar and ranged from 14 to 20 micromol/min/mg. nCMO and rCMO from the Escherichia coli expression system exhibited molecular masses, determined by electrospray mass spectrometry, of 60,800 and 61,615 Da, respectively, an increase for the recombinant enzyme equivalent to the mass of the His(6)-tag. However, rCMO expressed in Saccharomyces cerevisiae consistently exhibited a mass some 50 Da larger than rCMO expressed in bacteria. Edman degradation confirmed that rCMO purified from the E. coli system and nCMO shared the same N-terminal sequence, whereas no sequence information could be obtained for rCMO expressed in yeast. Therefore, the yeast-expressed enzyme possesses an additional posttranslational modification(s), possibly acylation, at the N-terminus. Expression in E. coli is the preferred system for future site-directed mutagenesis studies and crystallization efforts.     

Protein chemical and kinetic characterization of recombinant porcine ribonuclease inhibitor expressed in Saccharomyces cerevisiae

A cDNA encoding porcine ribonuclease inhibitor was used to express this protein in yeast under control of the PHO5 promoter. The recombinant protein was purified to homogeneity with a yield of 0.2 mg/g of yeast cells (wet weight) and was found to be indistinguishable from the inhibitor isolated from porcine liver on the basis of the following criteria: the amino acid composition, the number of free sulfhydryl groups, the molecular weight of the native and the denatured protein, peptide mapping, and amino acid sequence analysis of the N- and C-terminal regions of the protein. A simple method was developed for measuring accurately the slow, tight-biding kinetics of the inhibition of ribonuclease by ribonuclease inhibitor. From the dependence of the observed inhibition constant on the substrate concentration, it could be concluded that RI was competitive with the substrate UpA. The dependence of the observed association rate constant on the substrate concentration was consistent with a two-step mechanism in which the substrate only competed in the second (isomerization) step. The values for the inhibition constant for the inhibition of RNase by the recombinant inhibitor, 67 fM, the association rate constant, 1.5 x 10(8) M-1.s-1, and the dissociation rate constant, 8.3 x 10(-6) s-1, were in good agreement with those obtained for the porcine liver RNase inhibitor.     

Protease B from Saccharomyces cerevisiae. Purification and characterization.

Protease B has been isolated from Saccharomyces cerevisiae and purified in six steps as follows: autolysis of the yeast cells, ammonium sulfate fractionation, activation of the proteolytic enzymes, chromatography on DEAE-cellulose, chromatography on CM-cellulose and finally, a second chromatography on DEAE-cellulose. The preparation was shown to be homogeneous on polyacrylamide gels in the absence as well as in the presence of sodium dodecylsulfate. Furthermore, the molecular weight (43,000 daltons) and the isoelectric point (5.45) were in good agreement with earlier published values. The amino acid composition is reported. The absence of disulfide bonds in protease B has to be outlined. The amino acid residues of the protein have been found to be folded nearly quantitatively (at least 80%) in a beta-conformation as deduced from a circular dichroism study. Finally, the tryptophan residues (5 mol/mol protein) are largely buried in the hydrophobic core of the enzyme.     

Saccharomyces cerevisiae replication factor C. I. Purification and characterization of its ATPase activity.

Saccharomyces cerevisiae replication factor C (RF-C) was purified 25,000-fold from a protease-deficient strain of yeast. RF-C is a complex of 6 subunits of 130, 86, 41, 40, 37, and 27 kDa. None of the subunits are related through proteolysis or differential phosphorylation. The assay for RF-C used as a substrate single-stranded DNA binding protein-coated singly primed single-stranded mp 18 DNA. This DNA was poorly replicated by yeast DNA polymerase delta with or without its cofactor proliferating cell nuclear antigen (PCNA). In the presence of RF-C, however, replication of the template proceeded efficiently when both ATP and PCNA were present as well. Formation of this replication-proficient complex of DNA polymerase delta required an input of one to two molecules of PCNA per replicated DNA molecule. DNA polymerase epsilon also formed an ATP-dependent complex with PCNA and RF-C. RF-C has a DNA-dependent ATPase activity, equally active on single-stranded and primed single-stranded mp18 DNA. Addition of PCNA stimulated the ATPase of RF-C on primed but not on unprimed DNA, indicating that the increase in ATPase was due to PCNA-enhanced binding of RF-C to the primer terminus. Calf thymus PCNA also stimulated the ATPase activity of yeast RF-C and participated in holoenzyme formation with DNA polymerase delta. These results attest to the structural and functional homology between yeast and mammalian cells for these components of the replication machinery.     

Biochemical and structural characterization of recombinant copper-metallothionein from Saccharomyces cerevisiae.

Methods were developed for large-scale purification of recombinant Cu-metallothionein (Cu-MT) for structural investigations and the determination of Cu-binding stoichiometry. Cu-MT of Saccharomyces cerevisiae overexpressed in Escherichia coli was purified using a procedure based on ion exchange and gel filtration chromatography followed by reversed-phase HPLC. The purified protein was fully characterized by electrophoresis, amino acid analysis, atomic absorption spectroscopy and elemental analysis, and was shown to contain 10 +/- 2 Cu(I) per molecule of protein. Small angle X-ray scattering measurements yielded a radius of gyration of 1.2 nm for the recombinant protein, indicating a more extended structure in solution than that derived from the recent NMR data [Peterson, C.W., Narula, S.S. & Armitage, I.A. (1996) FEBS Lett. 379, 85-93].     

Purification and characterization of a recombinant G-protein-coupled receptor, Saccharomyces cerevisiae Ste2p, transiently expressed in HEK293 EBNA1 cells

The production of milligram quantities of purified, active, folded membrane protein from heterologous expression systems remains a general challenge due to intrinsically low expression levels, misfolding, and instability. Here we report the overexpression and purification of milligram quantities of functional Saccharomyces cerevisiae G-protein-coupled receptor, Ste2p, from transiently transfected human embryonic kidney 293 EBNA1 cells. Fluorescent microscopy indicates localization of Ste2p-GFP and Fc-Ste2p-GFP fusion receptors to the cell membrane. Up to 2 mg (approximately 10 pmol/million cells) of the Fc-Ste2p-GFP fusion and 1 mg of a Ste2p-Strep-TagII/(His)8-tagged version were purified per liter of culture following protein A-Sepharose and Talon metal affinity chromatography, respectively. Two distinct fluorescent labels, the hydrophobic 7-(diethylamino)-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM) and the more hydrophilic fluorescein-5-maleimide (FM), were individually attached to the C-terminus of the alpha-mating factor ligand by addition of a reactive cysteine residue to produce active fluorescent pheromones. In vitro fluorescent ligand binding assays demonstrated that a high percentage of the recombinant purified receptor is correctly folded and able to bind ligand. KD values of 34 +/- 3 and 300 +/- 20 nM were observed respectively for the CPM- and FM-labeled ligands. These results combined with blue-shifted emission peaks and loss of fluorescent quenching observed for both fluorescent-labeled Cys alpha-factors when bound to receptor support a model in which the C-terminus of the ligand is packed in a hydrophobic pocket at the interface between the transmembrane and extracellular loop domains. Overall, we present an efficient system for recombinant production of milligram quantities of purified Ste2p in a biologically active form with applications to future structure and functional studies.     

Purification and characterization of recombinant spinach acyl carrier protein I expressed in Escherichia coli

Expression of plant acyl carrier protein (ACP) in Escherichia coli at levels above that of constitutive E. coli ACP does not appear to substantially alter bacterial growth or fatty acid metabolism. The plant ACP expressed in E. coli contains pantetheine and approximately 50% is present in vivo as acyl-ACP. We have purified and characterized the recombinant spinach ACP-I. NH2-terminal amino acid sequencing indicated identity to authentic spinach ACP-I, and there was no evidence for terminal methionine or formylmethionine. Recombinant ACP-I was found to completely cross-react immunologically with polyclonal antibody raised to spinach ACP-I. Recombinant ACP-I was a poor substrate for E. coli fatty acid synthesis. In contrast, Brassica napus fatty acid synthetase gave similar reaction rates with both recombinant and E. coli ACP. Similarly, malonyl-coenzyme A:acyl carrier protein transacylase isolated from E. coli was only poorly able to utilize the recombinant ACP-I while the same enzyme from B. napus reacted equally well with either E. coli ACP or recombinant ACP-I. E. coli acyl-ACP synthetase showed a higher reaction rate for recombinant ACP-I than for E. coli ACP. Expression of spinach ACP-I in E. coli provides, for the first time, plant ACP in large quantities and should aid in both structural analysis of this protein and in investigations of the many ACP-dependent reactions of plant lipid metabolism.     

Purification and characterization of the hypoxanthine-guanine phosphoribosyltransferase from Saccharomyces cerevisiae.

1. Hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) from Saccharomyces cerevisiae was purified 9400-fold by affinity chromatography giving rise to an electrophoretically homogeneous preparation. 2. The molecular weight of the enzyme was determined by gel filtration with Sephadex G-100 and by sodium dodecylsulfate gel electrophoresis. Both methods reveal a molecular weight of 51,000. 3. The enzyme requires Mg2+ and has its pH optimum at 8.5. 4. Isoelectric focussing as well as gel electrophoresis of the purified extract reveals a single band which exhibits enzyme activity. The isoelectric point of the enzyme is 5.1. 5. The enzyme displays Michaelis-Menten kinetics with apparent Michaelis constants for hypoxanthine, guanine and phosphoribosylpyrophosphate of 23 microns, 18 microns, and 50 microns respectively.     

Purification and characterization of phosphatidylinositol kinase from Saccharomyces cerevisiae

The membrane-associated phospholipid biosynthetic enzyme phosphatidylinositol kinase (ATP:phosphatidylinositol 4-phosphotransferase, EC 2.7.1.67) was purified 8,000-fold from Saccharomyces cerevisiae. The purification procedure included Triton X-100 solubilization of microsomal membranes, DE-52 chromatography, hydroxylapatite chromatography, octyl-Sepharose chromatography, and two consecutive Mono Q chromatographies. The procedure resulted in the isolation of a protein with a subunit molecular weight of 35,000 that was 96% of homogeneity as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phosphatidylinositol kinase activity was associated with the purified Mr 35,000 subunit. Maximum phosphatidylinositol kinase activity was dependent on magnesium ions and Triton X-100 at pH 8. The true Km values for phosphatidylinositol and MgATP were 70 microM and 0.3 mM, and the true Vmax was 4,750 nmol/min/mg. The turnover number for the enzyme was 166 min-1. Results of kinetic and isotopic exchange reactions indicated that phosphatidylinositol kinase catalyzed a sequential Bi Bi reaction mechanism. The enzyme bound to phosphatidylinositol prior to ATP and phosphatidylinositol 4-phosphate was the first product released in the reaction. The equilibrium constant for the reaction indicated that the reverse reaction was favored in vitro. The activation energy for the reaction was 31.5 kcal/mol, and the enzyme was thermally labile above 30 degrees C. Phosphatidylinositol kinase activity was inhibited by calcium ions and thioreactive agents. Various nucleotides including adenosine and S-adenosylhomocysteine did not affect phosphatidylinositol kinase activity.     

Purification and characterization of recombinant porcine prorelaxin expressed in Escherichia coli.

In this report we describe the purification and characterization of recombinant porcine prorelaxin expressed in Escherichia coli. Nucleotide sequence encoding porcine prorelaxin was inserted into an E. coli expression vector, pOTS, and the recombinant plasmid was transformed into the E. coli host (AR120). Upon induction with nalidixic acid, the 19-kDa recombinant porcine prorelaxin was produced at a level of approximately 8% of the total accumulated cell protein. The recombinant prorelaxin was purified to homogeneity by CM-cellulose chromatography and reversed-phase HPLC, after refolding in the presence of reduced and oxidized glutathione and a low concentration of guanidine-HCl. The identity of the recombinant prorelaxin was confirmed by the correct size, immunoreactivity with antibodies against native porcine relaxin, and direct amino-terminal sequence analysis. Furthermore, the purified recombinant prorelaxin could be converted to the 6-kDa relaxin by limited digestion with trypsin. Trypsin was shown to cleave at the carboxyl side of Arg29 and Arg137 residues of the recombinant prorelaxin, producing the des-ArgA1-B29-relaxin, and degrade the 13-kDa connecting peptide into small peptides. Both the recombinant prorelaxin and converted relaxin were found to be biologically active in an in vitro bioassay for relaxin.     

Purification and characterization of Saccharomyces cerevisiae uridine monophosphate kinase

The SOC8 gene was isolated as an extragenic suppressor of cdc8 mutant cells. It has been suggested that SOC8 is allelic with the URA6 gene which was originally identified as a uridine monophosphate kinase. In this article, we describe the purification of the uridine monophosphate kinase from a yeast Saccharomyces cerevisae strain that overproduces the activity 8-fold. The protein was purified through Fast-Flow Q-Separose, phosphocellulose, blue-agarose, and fast protein liquid chromatography Superose 12 columns, and appears homogeneous by sodium dodecyl sulfate-polyacrylamide gel analysis. The uridine monophosphate kinase contains a single polypeptide with a molecular weight of 25,000, as evidence by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration analysis. The amino acid composition has also been determined. Substrate specificity studies show that the relative activity of nucleoside monophosphates is in order of UMP greater than dUMP, and to a lesser extent, dTMP, GMP, and dGMP. The Km and Vm of UMP, dUMP, and dTMP have been determined.     

Purification and characterization of CDP-diacylglycerol synthase from Saccharomyces cerevisiae

The membrane-associated phospholipid biosynthetic enzyme CDP-diacylglycerol synthase (CTP:phosphatidate cytidylyltransferase, EC 2.7.7.41) was purified 2,300-fold from Saccharomyces cerevisiae. The purification procedure included Triton X-100 solubilization of mitochondrial membranes, CDP-diacylglycerol-Sepharose affinity chromatography, and hydroxylapatite chromatography. The procedure resulted in a nearly homogeneous enzyme preparation as determined by native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radiation inactivation of mitochondrial associated and purified CDP-diacylglycerol synthase suggested that the molecular weight of the native enzyme was 114,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme preparation yielded two subunits with molecular weights of 56,000 and 54,000. Antibodies prepared against the purified enzyme immunoprecipitated CDP-diacylglycerol synthase activity and subunits. CDP-diacylglycerol synthase activity was dependent on magnesium ions and Triton X-100 at pH 6.5. Thio-reactive agents inhibited activity. The activation energy for the reaction was 9 kcal/mol, and the enzyme was thermally labile above 30 degrees C. The Km values for CTP and phosphatidate were 1 and 0.5 mM, respectively, and the Vmax was 4,700 nmol/min/mg. Results of kinetic and isotopic exchange reactions suggested that the enzyme catalyzes a sequential Bi Bi reaction mechanism.     

Purification and characterization of phosphatidate phosphatase from Saccharomyces cerevisiae

Membrane-associated phosphatidate phosphatase (EC 3.1.3.4) was purified 9833-fold from the yeast Saccharomyces cerevisiae. The purification procedure included sodium cholate solubilization of total membranes followed by chromatography with DE53, Affi-Gel Blue, hydroxylapatite, Mono Q, and Superose 12. The procedure resulted in the isolation of a protein with a subunit molecular weight of 91,000 that was apparently homogeneous as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phosphatidate phosphatase activity was associated with the purified 91,000 subunit. The molecular weight of the native enzyme was estimated to be 93,000 by gel filtration chromatography with Superose 12. Maximum phosphatidate phosphatase activity was dependent on magnesium ions and Triton X-100 at pH 7. The Km value for phosphatidate was 50 microM, and the Vmax was 30 mumol/min/mg. The turnover number (molecular activity) for the enzyme was 2.7 x 10(3) min-1 at pH 7 and 30 degrees C. The activation energy for the reaction was 11.9 kcal/mol, and the enzyme was labile above 30 degrees C. Phosphatidate phosphatase activity was sensitive to thioreactive agents. Activity was inhibited by the phospholipid intermediate CDP-diacylglycerol and the neutral lipids diacylglycerol and triacylglycerol.