Spectral and enzymatic properties of human recombinant myeloperoxidase: comparison with the mature enzyme.

Human recombinant myeloperoxidase (recMPO), purified from an engineered Chinese hamster ovary (CHO) cell line, has been characterized and compared to the mature enzyme isolated from polymorphonuclear leukocytes. Both molecules appear essentially similar in physicochemical enzymatic terms according to the following observations. 1. The unprocessed recombinant protein displays the characteristic light absorption spectra of ferric mature MPO and exhibits its typical spectral changes in the presence of dithionite or hydrogen peroxide. 2. The addition of 14C-labeled 5-aminolevulinic acid, a heme precursor, to the culture medium of recombinant CHO cells yields labeled recMPO, indicating the presence of a heme-like structure in the molecule. 3. Like mature MPO, recMPO has a peroxidatic activity and catalyzes the oxidation of chloride ions in the presence of hydrogen peroxide, producing hypochlorous acid as measured by the monochlorodimedon assay. For both enzymes, the chlorinating activity optimally occurs around pH 5.0 at about 100 microM of hydrogen peroxide and is strongly inhibited by methimazole. 4. Diethylpyrocarbonate significantly reduces the enzymatic activity of both molecules, suggesting that histidine residues may be of prime importance in the active site of the enzymes. 5. According to infrared spectroscopy data, both enzymes present a very similar secondary structure organization. In conclusion, the data suggest that the processing of the precursor enzyme (recMPO) into the mature form occurs without major structural and functional consequences.     

Rat liver beta-glucuronidase. cDNA cloning, sequence comparisons and expression of a chimeric protein in COS cells.

A cDNA for rat liver beta-glucuronidase was isolated, its sequence determined and its expression after transfection into COS cells studied. The deduced amino acid sequence of the rat liver clone showed 77% homology with that from the cDNA for human placental beta-glucuronidase and 47% homology with that deduced from the cDNA for Escherichia coli beta-glucuronidase. Several differences were found between the cDNA from rat liver and that previously reported from rat preputial gland. Only one change leads to an amino acid difference in the mature enzyme. A chimeric clone was constructed by using a fragment encoding the first 18 amino acid residues of the signal sequence from the human placental cDNA clone and a fragment from the rat clone encoding four amino acid residues of the signal sequence, all 626 amino acid residues of the mature rat enzyme, and all of the 3' untranslated region. After transfection into COS cells the chimeric clone expressed beta-glucuronidase activity that was specifically immunoprecipitated by antibody to rat beta-glucuronidase. The Mr value of 76,000 of the expressed gene product was characteristic of the glycosylated rat enzyme. It was proteolytically processed in COS cells to Mr 75,000 6 h after metabolic labelling. At least 50% of the expressed enzyme was secreted at 60 h post-transfection, but the secreted enzyme did not undergo proteolytic processing. These results provide evidence that the partial cDNA isolated from a rat liver library contains the complete coding sequence for the mature rat liver enzyme and that the chimeric signal sequence allows normal biosynthesis and processing of the transfected rat liver enzyme in COS cells.     

Primary structure of rat brain prostaglandin D synthetase deduced from cDNA sequence

The amino acid sequence of rat brain prostaglandin D synthetase (Urade, Y., Fujimoto, N., and Hayaishi, O. (1985) J. Biol. Chem. 260, 12410-12415) was determined by a combination of cDNA and protein sequencing. cDNA clones specific for this enzyme were isolated from a lambda gt11 rat brain cDNA expression library. Nucleotide sequence analyses of cloned cDNA inserts revealed that this enzyme consisted of a 564- or 549-base pair open reading frame coding for a 188- or 183-amino acid polypeptide with a Mr of 21,232 or 20,749 starting at the first or second ATG. About 60% of the deduced amino acid sequence was confirmed by partial amino acid sequencing of tryptic peptides of the purified enzyme. The recognition sequence for N-glycosylation was seen at two positions of amino acid residues 51-53 (-Asn-Ser-Ser-) and 78-80 (-Asn-Leu-Thr-) counted from the first Met. Both sites were considered to be glycosylated with carbohydrate chains of Mr 3,000, since two smaller proteins with Mr 23,000 and 20,000 were found during deglycosylation of the purified enzyme (Mr 26,000) with N-glycanase. The prostaglandin D synthetase activity was detected in fusion proteins obtained from lysogens with recombinants coding from 34 and 19 nucleotides upstream and 47 and 77 downstream from the first ATG, indicating that the glycosyl chain and about 20 amino acid residues of N terminus were not essential for the enzyme activity. The amino acid composition of the purified enzyme indicated that about 20 residues of hydrophobic amino acids of the N terminus are post-translationally deleted, probably as a signal peptide. These results, together with the immunocytochemical localization of this enzyme to rough-surfaced endoplasmic reticulum and other nuclear membrane of oligodendrocytes (Urade, Y., Fujimoto, N., Kaneko, T., Konishi, A., Mizuno, N., and Hayaishi, O. (1987) J. Biol. Chem. 262, 15132-15136) suggest that this enzyme is a membrane-associated protein.     

The hypoxanthine-guanine phosphoribosyltransferase of Schistosoma mansoni. Further characterization and gene expression in Escherichia coli

Due to the lack of de novo purine nucleotide biosynthesis, hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) is an essential enzyme in the human parasite Schistosoma mansoni for supplying guanine nucleotides and has been proposed as a potential target for antiparasitic chemotherapy. While the enzyme can be purified from adult schistosome worms, yields are too low to allow extensive structural and kinetic studies. We therefore cloned and sequenced the cDNA and gene encoding the schistosomal enzyme but were unable to positively identify the amino-terminal sequence of the enzyme from the DNA sequence. Knowledge of the exact amino terminus was necessary before accurate expression of active enzyme could be attempted. Therefore, we purified the HGPRTase from crude extracts of the adult worms. The purified enzyme has a subunit molecular mass of 26 kDa and an amino-terminal sequence of Met-Ser-Ser-Asn-Met. This sequence matched one of the potential initiation sites predicted from the cDNA and gene sequence. We next expressed the correct size cDNA of the S. mansoni HGPRTase in Escherichia coli using a vector that is regulated by a bacterial alkaline phosphatase promoter and uses an E. coli signal peptide for secretion of expressed product into the periplasmic space. Using this expression system, some of the recombinant enzyme is secreted and found to have a correct amino terminus. That remaining in the cytoplasm has part of the signal peptide attached to the amino terminus. The recombinant schistosomal HGPRTase isolated from the periplasm of the transformed E. coli was purified and found to have kinetic and physical properties identical to those of the native enzyme.     

Sequence and expression of Thai Rosewood beta-glucosidase/beta-fucosidase, a family 1 glycosyl hydrolase glycoprotein

Dalcochinin-8'-O-beta-glucoside beta-glucosidase (dalcochinase) from the Thai rosewood (Dalbergia cochinchinensis Pierre) has aglycone specificity for isoflavonoids and can hydrolyze both beta-glucosides and beta-fucosides. To determine its structure and evolutionary lineage, the sequence of the enzyme was determined by peptide sequencing followed by PCR cloning. The cDNA included a reading frame coding for 547 amino acids including a 23 amino acid propeptide and a 524 amino acid mature protein. The sequences determined at peptide level were found in the cDNA sequence, indicating the sequence obtained was indeed the dalcochinase enzyme. The mature enzyme is 60% identical to the cyanogenic beta-glucosidase from white clover glycosyl hydrolase family 1, for which an X-ray crystal structure has been solved. Based on this homology, residues which may contribute to the different substrate specificities of the two enzymes were identified. Eight putative glycosylation sites were identified, and one was confirmed to be glycosylated by Edman degradation and mass spectrometry. The protein was expressed as a prepro-alpha-mating factor fusion in Pichia pastoris, and the activity of the secreted enzyme was characterized. The recombinant enzyme and the enzyme purified from seeds showed the same K(m) for pNP-glucoside and pNP-fucoside, had the same ratio of V(max) for these substrates, and similarly hydrolyzed the natural substrate, dalcochinin-8'-beta-glucoside.     

Nucleotide sequence for cDNA of bovine mitochondrial ATP-dependent protease and determination of N-terminus of the mature enzyme from the adrenal cortex.

We have determined the cDNA sequence encoding bovine mitochondrial ATP-dependent Lon protease. Since the 5'-end region of the cDNA was highly GC-rich and thus could not be amplified by the 5'-RACE method, a genomic DNA fragment containing an in-frame ATG was isolated and sequenced. The translated amino acid sequence contained 961 amino acids with a calculated molecular weight 106,665. Sequence similarities of the bovine enzyme to human and E. coli orthologs were 92 and 27%, respectively. The N-terminal amino acid sequence seemed to be a mitochondrial targeting signal. To determine the cleavage site of the signal sequence we analyzed the mature enzyme purified from bovine adrenocortical mitochondria. Analysis of CNBr-digested peptides revealed that the N-terminus was heterogeneous. We suggest that nonspecific aminopeptidase might remove several amino acids from the N-terminus after mitochondrial processing peptidase has cleaved Gly(67)-Leu(68) or Leu(68)-Trp(69).     

Cloning and expression in Pichia pastoris of an Irpex lacteus rhamnogalacturonan hydrolase tolerant to acetylated rhamnogalacturonan

In order to produce a recombinant rhamnogalacturonase from the basidiomycete Irpex lacteus using a molecular approach, PCR primers were designed based on a sequence alignment of four known ascomycete rhamnogalacturonases. Using 5' and 3' rapid amplification of cDNA ends (RACE) experiments, a 1,437-bp full-length cDNA containing an open reading frame of 1,329 bp was isolated. The corresponding putative protein sequence is of 443 amino acids and contains a secretion signal sequence of 22 amino acids. The theoretical mass of this protein is 44.6 kDa with a theoretical isoelectric point of 6.2. The amino acid sequence shared not only significant identities with ascomycete and basidiomycete putative rhamnogalacturonases but also complete similarity with peptides obtained from a recently purified rhamnogalacturonase from I. lacteus. The recombinant protein was successfully expressed in active form in Pichia pastoris. SDS-PAGE assay demonstrated that the recombinant enzyme was secreted in the culture medium and had a molar mass of 56 kDa. This recombinant rhamnogalacturonan hydrolase exhibited a pH optimum between 4.5 and 5 and a temperature optimum between 40°C and 50°C, which correspond to that of the native rhamnogalacturonase from I. lacteus. The study of its specificity through reaction products analysis showed that it was highly tolerant to the presence of acetyl groups on its substrate, even more than the native enzyme.     

Cloning, expression, and characterization of a cDNA encoding snake venom metalloprotease

A cDNA clone, MT-c, encoding metalloprotease was isolated from snake (Agkistrodon halys brevicadus) venom gland cDNA library. Deduced amino acid sequence indicated that MT-c is composed of a signal sequence, amino-terminal propeptide, a central metalloprotease domain, and a Lys-Gly-Asp (KGD) disintegrin domain. The partial cDNA encoding metalloprotease and disintegrin domain was subcloned and expressed in E. coli. The expressed MT-c protein was purified and successfully refolded into functional form retaining the enzyme activity. Analyses of the purified recombinant protease activity revealed that the enzyme hydrolyzes extracellular matrix proteins including type I gelatin, type IV and type V collagen, while type I, II, III collagens and fibronectin were insensitive to the proteolytic digestion. The recombinant enzyme was also able to degrade fibrinogen by specifically cleaving A alpha chain of the protein.     

Molecular cloning and functional analysis of apoxin I, a snake venom-derived apoptosis-inducing factor with L-amino acid oxidase activity

We previously purified apoxin I, an apoptosis-inducing factor with L-amino acid oxidase (LAO) activity, from Western diamondback rattlesnake venom. To determine the primary structure of apoxin I, we cloned its cDNA. The amino acid sequence showed that apoxin I has an FAD binding domain and shares homology with L-amino acid oxidase (LAO) from Neurospora crassa, human monoamine oxidase B, and mouse interleukin 4-induced F1G1 protein. The full-length apoxin I has an N-terminal signal sequence that is processed in mature apoxin I in venom. When the apoxin I gene was transfected into human 293T cells, the recombinant protein was expressed in the cells, and a significant amount of apoxin I was secreted into the medium. The secreted recombinant apoxin I protein showed LAO and apoptosis-inducing activity, but the recombinant protein in the cells did not, suggesting that maturation and secretion of the apoxin I protein is needed for its activity. Treating the transfected cells with tunicamycin inhibited the secretion and LAO activity of the recombinant apoxin I. In addition, deleting the amino-terminal region flanking the signal sequence, the FAD-binding domain and the carboxy-terminal region abolished the secretion and LAO activity of the recombinant proteins. These results indicate that in order for apoxin I to become active, these regions and posttranslational modification, such as N-glycosylation, are required.     

A transient kinetic study on the reactivity of recombinant unprocessed monomeric myeloperoxidase

Spectral and kinetic features of the redox intermediates of human recombinant unprocessed monomeric myeloperoxidase (recMPO), purified from an engineered Chinese hamster ovary cell line, were studied by the multi-mixing stopped-flow technique. Both the ferric protein and compounds I and II showed essentially the same kinetic behavior as the mature dimeric protein (MPO) isolated from polymorphonuclear leukocytes. Firstly, hydrogen peroxide mediated both oxidation of ferric recMPO to compound I (1.9 x 10(7) M(-1) s(-1), pH 7 and 15 degrees C) and reduction of compound I to compound II (3.0 x 10(4) M(-1) s(-1), pH 7 and 15 degrees C). With chloride, bromide, iodide and thiocyanate compound I was reduced back to the ferric enzyme (3.6 x 10(4) M(-1) s(-1), 1.4 x 10(6) M(-1) s(-1), 1.4 x 10(7) M(-1) s(-1) and 1.4 x 10(7) M(-1) s(-1), respectively), whereas the endogenous one-electron donor ascorbate mediated transformation of compound I to compound II (2.3 x 10(5) M(-1) s(-1)) and of compound II back to the resting enzyme (5.0 x 10(3) M(-1) s(-1)). Comparing the data of this study with those known from the mature enzyme strongly suggests that the processing of the precursor enzyme (recMPO) into the mature form occurs without structural changes at the active site and that the subunits in the mature dimeric enzyme work independently.     

Juvenile hormone esterase (JHE) from Tenebrio molitor: full-length cDNA sequence,in vitro expression,and characterization of the recombinant protein

Juvenile hormone regulates the development and reproduction in a variety of insects. Juvenile hormone esterase (JHE) is a selective enzyme, which hydrolyzes the methyl ester of JH and alters its activity. In Tenebrio molitor, JHE has been previously purified from pupae and a partial cDNA was amplified by RT-PCR using fat body mRNA. The previous report indicated that several forms of the JHE protein were present in pupal homogenate. In this study, we report the full-length cDNA, which was obtained by RACE methods. The deduced protein sequence corresponds to peptides from two proteins of different molecular weights in the previous study. The coding region of the full-length cDNA was subcloned into the AcMNPV genome and high levels of expression of the JHE enzyme from the viral p10 promoter were demonstrated in cell culture. The majority of JHE is secreted from the cells as a soluble enzyme. The recombinant JHE enzyme was biochemically characterized. The recombinant protein appears by PAGE analysis as a monomer of approximately the same MW (66000) and pI (4.9) as was expected from the deduced amino acid sequence of the cDNA.     

Expression of functional recombinant antibody molecules in insect cell expression systems

Recombinant single-chain variable-fragment molecules (scFv) were constructed from a cell line expressing a monoclonal antibody against African cassava mosaic virus (ACMV) and expressed in Escherichia coli. DNA sequences that encoded the scFv were manipulated to allow scFv expression in insect cell lines. A recombinant baculovirus containing the scFv cDNA was constructed and large amounts of scFv were produced in each of three insect cell lines infected with the baculovirus. However, the scFv were not secreted into the medium by any of the cell lines despite the scFv having been linked to a honeybee melittin leader sequence. The same scFv cDNA construct was introduced into Drosophila DS2 cells and a stable recombinant cell line was obtained that produced scFv that was secreted into the medium. Culture medium containing the scFv was used directly in enzyme-linked immunosorbent assay (ELISA) tests to detect ACMV in plant tissues. Another construct that encoded the Ckappa domain of human IgG was fused to the C-terminus of the scFv that was produced and expressed in Drosophila cells. This scFv derivative also accumulated in the medium and was more active in ELISA than scFv lacking the Ckappa domain.     

嗜热真菌Thermomyces lanuginosus热稳定几丁质酶基因的cDNA克隆及表达

根据Thermomyces lanuginosus热稳定几丁质酶Chit的N-端氨基酸序列和同源保守序列设计简并引物,通过RT-PCR及快速扩增cDNA末端(RACE)的方法,克隆了该几丁质酶的编码基因chit,全长cDNA为1500bp,包含一个由442个氨基酸组成的开放阅读框。该基因已在GenBank中注册,登录号为DQ092332。将成熟肽几丁质酶Chit阅读框与酵母表达载体pPIC9K连接,构建重组质粒pPIC9K/chit,转化毕赤酵母GS115,在甲醇的诱导下,成功地分泌出具生物活性的几丁质酶,诱导6d后酶活性达2.261U/mL,酶蛋白表达量为0.36mg/mL。该酶的最适反应温度和pH值分别为60℃和5.5,该酶在50℃以下稳定;65℃的半衰期为40min。     

A catalase from the freshwater mussel Cristaria plicata with cloning, identification and protein characterization

Catalase is an important antioxidant protein which can protect organisms against various oxidative stresses by eliminating hydrogen peroxide. The catalase cDNA of Cristaria plicata (cpCAT) was cloned from the haemocytes using degenerate primers by the method of 3' and 5' rapid amplification of cDNA ends PCR. The gene is 4863 bp long and has a total of two introns and three exons. The precise size and location of the introns and exons have been determined. In addition the full-length cDNA of cpCAT contained 2618 bp, The cDNA contained a 5' untranslated region (UTR) of 136 nucleotides, the 3' UTR of 979 bp with a canonical polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) of 1503 bp, encoding 501 amino acid residues with 56.86 kDa predicted molecular weight. The theoretical isoelectric point was 6.77. BLAST analysis showed that the deduced amino acid sequence of cpCAT had significant homology to catalases from animals, plants and bacteria. The deduced amino acid sequence of cpCAT had characteristic features of catalase family such as catalytic site motif (61FNRERIPERVVHAKGAG77), heme-ligand signature motif (351RLYSYSDTH359), two glycosylation sites (N145, N436), NADPH binding site and the three catalytic amino acid residues (His72, Asn145 and Tyr355). It had no signal peptide. The phylogenetic tree indicated that cpCAT gene was very close to the gene of scallops, Chlamys farreri. The enzymatic activity of purified recombinant cpCAT was 11194.4 ± 40.4 U/mg, it might resist against H(2)O(2). The recombinant enzyme held higher thermal stability, the optimum temperature was 25 °C, it retained more than 82% activity between 25 and 60 °C. The stability of the recombinant enzyme were higher between pH 5 and 10, and the optimal pH value was 7.0. When cpCAT was treated with 2-4 moL/L urea and 1%-3% SDS, the activity was also stable, it kept more than 80% activity.     

Expression of human DNA polymerase beta in Escherichia coli and characterization of the recombinant enzyme

The coding region of a human beta-polymerase cDNA, predicting a 335 amino acid protein, was subcloned in the Escherichia coli expression plasmid pRC23. After induction of transformed cells, the crude soluble extract was found to contain a new protein immunoreactive with beta-polymerase antibody and corresponding in size to the protein deduced from the cDNA. This protein was purified in a yield of 1-2 mg/50 g of cells. The recombinant protein had about the same DNA polymerase specific activity as beta-polymerase purified from mammalian tissues, and template-primer specificity and immunological properties of the recombinant polymerase were similar to those of natural beta-polymerases. The purified enzyme was free of nuclease activity. We studied detailed catalytic properties of the recombinant beta-polymerase using defined template-primer systems. The results indicate that this beta-polymerase is essentially identical with natural beta-polymerases. The recombinant enzyme is distributive in mode of synthesis and is capable of detecting changes in the integrity of the single-stranded template, such as methylated bases and double-stranded region. The enzyme recognizes a template region four to seven bases downstream of the primer 3' end and utilizes alternative primers if this downstream template region is double stranded. The enzyme is unable to synthesize past methylated bases N3-methyl-dT or O6-methyl-dG.     

Porcine pyridoxal kinase: c-DNA cloning, expression and confirmation of its primary sequence

Porcine brain pyridoxal kinase has been cloned. A 1.2 kilo-based cDNA with a 966-base pair open reading frame was determined from a porcine brain cortex cDNA library using PCR technique. The DNA sequence was shown to encode a protein of 322 amino acid residues with a molecular mass of 35.4 kDa. The amino acid sequence deduced from the nucleotide sequence of the cDNA was shown to match the partial primary sequence of pyridoxal kinase. Expression of the cloned cDNA in E. coli has produced a protein which displays both pyridoxal kinase activity and immunoreactivity with monoclonal antibodies raised against natural enzyme from porcine brain. With respect to the physical properties, it is shown that the recombinant protein exhibits identical kinetic parameters with the pure enzyme from porcine brain. Although the primary sequence of porcine pyridoxal kinase has been shown to share 87% homology with the human enzyme, we have shown that the porcine enzyme carries an extra peptide of ten amino acid residues at the N-terminal domain.     

Isolation and characterization of the gene encoding gluconolactonase from Zymomonas mobilis.

The gene encoding the enzyme gluconolactonase (D-glucono-delta-lactone lactonohydrolase, EC 3.1.1.17) has been isolated from a recombinant library of genomic Zymomonas mobilis DNA, by detection of enzyme activity in recombinant clones. The gene encoded a protein of 320 amino acids, which is processed to the mature enzyme of 285 amino acids (31079 Da) by cleavage at an Ala-Ala bond, as determined from N-terminal sequencing of the purified enzyme. A minor sequence commencing at amino acid 6 is suggestive of an alternative start of translation at the ATG codon of amino acid 5; in this case the expressed enzyme would remain cytoplasmic, whereas it is presumed that the main portion is directed to the membrane of periplasm by the leader sequence.     

Isolation and characterization of an unprocessed extracellular myeloperoxidase in HL-60 cell cultures

Extracellular myeloperoxidase of human myeloid leukemia HL-60 cells was purified to homogeneity from its culture supernatant by ammonium sulfate fractionation, CM-Sepharose column chromatography, and monoclonal antibody-Sepharose affinity column chromatography. The yield of enzyme activity was 38% that of the ammonium sulfate fraction. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified preparation gave a single band of approximately 84 kDa. Analysis of protein blot with antibodies specific for the light and heavy chains of myeloperoxidase indicated that the enzyme contained a light and a heavy chain in a single polypeptide. The amino-terminal amino acid sequence of the enzyme began at amino acid residue 155 of the 745-amino acid sequence predicted from myeloperoxidase cDNA, indicating that the enzyme consisted of 591 amino acids. Sucrose density gradient centrifugation of the enzyme showed that the enzyme was a monomeric form. In pulse-chase experiments on HL-60 cells with [35S]methionine, pulse-labeled myeloperoxidase precursors were shown to be processed to a light chain and a heavy chain of cellular enzyme. During a 3-day chase period, newly formed processed monomeric enzyme was converted to a dimeric form.     

Cloning and expression of the Gal beta 1, 3GalNAc alpha 2,3-sialyltransferase.

Sequence information obtained by NH2-terminal sequence analysis of two molecular weight forms (45 and 48 kDa) of the porcine Gal beta 1,3GalNAc alpha 2,3-sialyltransferase was used to clone a full-length cDNA of the enzyme. The cDNA sequence revealed an open reading frame coding for 343 amino acids and a putative domain structure consisting of a short NH2-terminal cytoplasmic domain, a signal-anchor sequence, and a large COOH-terminal catalytic domain. This domain structure was confirmed by construction of a recombinant sialyltransferase in which the cytoplasmic domain and signal-anchor sequence of the enzyme was replaced with the cDNA of insulin signal sequence. Expression of the resulting construct in COS-1 cells produced an active sialyltransferase which was secreted into the medium in soluble form. Comparison of the cDNA sequence of the sialyltransferase with GenBank produced no significant homologies except with the previously described Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase. Although the cDNA sequences of these two enzymes were largely nonhomologous, there was a 45-amino acid sequence which exhibited 65% identity. This observation suggests that the two sialyltransferases were derived, in part, from a common gene.