Purification and cDNA cloning of rat 6-pyruvoyl-tetrahydropterin synthase

6-Pyruvoyl-tetrahydropterin synthase, which catalyzes the second step in the biosynthesis of tetrahydrobiopterin, was purified approximately 18,000-fold to apparent homogeneity from rat liver. The molecular mass of the native enzyme was estimated to be 83 kDa by gel filtration. The enzyme showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to a molecular mass of 17 kDa. Up to 24 residues of the NH2-terminal sequence were determined by Edman degradation, which released a single amino acid at each step. These results indicate that the enzyme consists of identical subunits. The purified enzyme was digested with lysyl endopeptidase or V8 protease, and 11 peptide fragments were isolated. On the basis of the sequences of these peptides, oligonucleotides were synthesized and used to screen a rat liver cDNA library, and one cDNA clone was isolated. The complete nucleotide sequence of the 1176-base pair cDNA was then determined. The deduced amino acid sequence contained 144 amino acid residues, but a NH2-terminal four-amino acid sequence was not found in the purified protein. Therefore, the mature protein consists of 140 amino acids. A single mRNA band of 1.3 kilobases was obtained by RNA blot analysis of rat liver. The predicted amino acid sequence of 6-pyruvoyl-tetrahydropterin synthase was compared with the Protein Sequence Database of the National Biomedical Research Foundation, revealing significant local similarity to large T antigens from the polyomavirus family.     

Primary structure of rat liver mannan-binding protein deduced from its cDNA sequence

cDNA clones encoding rat liver mannan-binding protein (MBP), a lectin specific for mannose and N-acetylglucosamine, were isolated from a rat liver cDNA library carried in lambda gt 11, by screening with affinity purified polyclonal rabbit anti-rat liver MBP antibodies. The nucleotide sequence of the cDNA determined by the dideoxy method revealed the complete amino acid sequence of the MBP (226 residues). The NH2-terminal residue of the MBP, glutamic acid, was preceded by a predominantly hydrophobic stretch of 18 amino acids, which was assumed to be a signal peptide. Near the NH2-terminal, there was a collagen-like domain, which consisted of 19 repeats of the sequence Gly-X-Y. Here, X and Y were frequently proline and lysine. Three proline and lysine residues were hydroxylated, and one of the latter appeared to link to galactose. Computer analysis of several lectins for sequence homology suggested that the COOH-terminal quarter of the MBP is associated with the calcium binding as well as carbohydrate recognition.     

Cloning, characterization, and expression in Escherichia coli of a gene encoding Listeria seeligeri catalase, a bacterial enzyme highly homologous to mammalian catalases.

A gene coding for catalase (hydrogen-peroxide:hydrogen-peroxide oxidoreductase; EC 1.11.1.6) of the gram-positive bacterium Listeria seeligeri was cloned from a plasmid library of EcoRI-digested chromosomal DNA, with Escherichia coli DH5 alpha as a host. The recombinant catalase was expressed in E. coli to an enzymatic activity approximately 50 times that of the combined E. coli catalases. The nucleotide sequence was determined, and the deduced amino acid sequence revealed 43.2% amino acid sequence identity between bovine liver catalase and L. seeligeri catalase. Most of the amino acid residues which are involved in catalytic activity, the formation of the active center accession channel, and heme binding in bovine liver catalase were also present in L. seeligeri catalase at the corresponding positions. The recombinant protein contained 488 amino acid residues and had a calculated molecular weight of 55,869. The predicted isoelectric point was 5.0. Enzymatic and genetic analyses showed that there is most probably a single catalase of this type in L. seeligeri. A perfect 21-bp inverted repeat, which was highly homologous to previously reported binding sequences of the Fur (ferric uptake regulon) protein of E. coli, was detected next to the putative promoter region of the L. seeligeri catalase gene.     

Rat aldolase isozyme gene

Rat aldolase B mRNA was partially purified from liver polysomes by an immunochemical technique followed by oligo(dT)-cellulose column chromatography. Double-stranded cDNA, synthesized from this mRNA, was inserted into the PstI site of plasmid pBR322 employing the oligo(dC)-oligo(dG) tailing method. Clones containing aldolase B cDNA inserts were selected by colony hybridization using 32P-labeled purified mRNA as a specific probe. Several recombinant plasmids containing 600 to 1000 base pair inserts were isolated. Hybrid selection-translation experiments showed that they hybridize specifically with aldolase B mRNA. By overlapping restriction maps of several individual cDNA inserts, it was found that they spanned 1200 base pairs, which represented about 70% of the aldolase B mRNA sequence. The nucleotide sequence of the cDNA was then determined and the sequence of 180 amino acids from the COOH terminus and the entire 3' untranslatable nucleotide sequence were clarified. Although the complete amino acid sequence of rat aldolase B has not yet been reported, it was found that several amino acids neighboring the COOH-terminal tyrosine obtained by carboxypeptidase digestion completely coincided with those determined from the cDNA sequence; i.e. -Ser-Leu-Phe-Thr-Ala-Ser-Tyr-Thr-Tyr. Furthermore, a putative active site peptide appeared and is extensively homologous to those of rabbit aldolases A and B.     

Cloning and recombinant expression of the La RNA-binding protein from Trypanosoma brucei

We report the isolation, cloning and recombinant expression of a Trypanosoma brucei homolog of the La RNA-binding protein. Based on peptide sequence information we have isolated a cDNA clone which encodes a protein of 335 amino acids with a predicted molecular weight of 37.7 kDa. The amino acid sequence fits the domain structure of known La proteins and contains a putative ATP-binding site located in the COOH-terminal domain. The cDNA was expressed as a glutathione S-transferase fusion protein in Escherichia coli, and the recombinant protein displayed RNA-binding activity in an electrophoretic mobility shift assay.     

Molecular cloning of a cDNA encoding chicken T-protein of the glycine cleavage system and expression of the functional protein in Escherichia coli. Effect of mRNA secondary structure in the translational initiation region on expression.

DNA clones encoding chicken T-protein of the glycine cleavage system were isolated from chicken liver lambda gt10 cDNA libraries. Three overlapping clones provided an open reading frame of 1176 nucleotides that predicts a polypeptide of 392 amino acids (M(r) 42,056) comprised of a 16-residue mitochondrial targeting sequence and a 376-residue mature protein (M(r) 40,292). The amino acid sequence predicted for the mature protein showed 67% identity with that of bovine T-protein. A cDNA encoding mature T-protein was constructed, and the nucleotide sequence just downstream of the initiation codon was modified without amino acid substitution to reduce the free energy of formation for the folded mRNA. Expression plasmids containing these cDNA variants produced large amounts of T-protein in Escherichia coli, while very low expression was observed with a plasmid containing wild type cDNA. Enzymatically active T-protein was obtained when the expression was conducted at 30 degrees C with 25 microM isopropyl-1-thio-beta-D-galactopyranoside. Under the full inducing condition (at 37 degrees C and 1 mM inducer), the expressed T-protein was recovered as insoluble and inactive protein. The recombinant T-protein was purified to near homogeneity with a yield of about 30%. Apparent molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is approximately 40,000, similar to the size of T-protein purified from chicken liver. NH2-terminal amino acid sequence analysis (9 residues) revealed 100% identity with chicken T-protein determined chemically. The kinetic properties of the recombinant T-protein resembled those of the native chicken T-protein.     

Molecular cloning of a cDNA for alpha-subunit of rat liver electron transfer flavoprotein

Two cDNA clones for the alpha-subunit of rat liver electron transfer flavoprotein were isolated and their nucleotide sequences were determined. The longer cDNA contained a protein-coding region of 900 nucleotides and 3'-noncoding region of 335 nucleotides. The identity of the clone was confirmed by matching the amino acid sequence predicted from the cDNA with the sequence of one of the lysyl endopeptidase-digested peptides from the purified alpha-subunit. The molecular weight of the protein calculated from the protein-coding nucleotides was approx. 3,000 daltons smaller than that of the precursor, suggesting that the cDNA was not of full length. The derived amino acid composition fairly agreed with the chemically determined amino acid composition of the purified alpha-subunit, indicating that the protein-coding region contains most of the mature alpha-subunit.     

Molecular cloning of cDNA for rat liver catalase

For the studies on the induction of peroxisomal enzymes by hypolipidemic agents, we have tried to isolate a cDNA clone for rat liver catalase. A recombinant clone, pMJ501, was isolated, of which cDNA insert specifically hybridized to catalase mRNA in hybridization-selected translation. On RNA blot hybridization, it hybridized to 2.4-kilobases RNA which was increased about 1.5-fold by the administration of di-(2-ethylhexyl)phthalate to the rats. The nucleotide sequence of the cDNA contains a reading frame for 109 amino acid residues which match the reported amino acid sequence of bovine liver catalase at the carboxyl end with 82% homology. It is concluded that pMJ501 contains a cDNA sequence for rat liver catalase.     

cDNA and deduced amino acid sequence of a mouse DNA repair enzyme (APEX nuclease) with significant homology to Escherichia coli exonuclease III

We purified a mouse DNA repair enzyme having apurinic/apyrimidinic endonuclease, DNA 3'-phosphatase, 3'-5'-exonuclease and DNA 3' repair diesterase activities, and designated the enzyme as APEX nuclease. A cDNA clone for the enzyme was isolated from a mouse spleen cDNA library using probes of degenerate oligonucleotides deduced from the N-terminal amino acid sequence of the enzyme. The complete nucleotide sequence of the cDNA (1.3 kilobases) was determined. Northern hybridization using this cDNA showed that the size of its mRNA is about 1.5 kilobases. The complete amino acid sequence for the enzyme predicted from the nucleotide sequence of the cDNA (APEX nuclease cDNA) indicates that the enzyme consists of 316 amino acids with a calculated molecular weight of 35,400. The predicted sequence contains the partial amino acid sequences determined by a protein sequencer from the purified enzyme. The coding sequence of APEX nuclease was cloned into pUC18 SmaI and HindIII sites in the control frame of the lacZ promoter. The construct was introduced into BW2001 (xth-11, nfo-2) strain cells of Escherichia coli. The transformed cells expressed a 36.4-kDa polypeptide (the 316 amino acid sequence of APEX nuclease headed by the N-terminal decapeptide of beta-galactosidase) and were less sensitive to methyl methanesulfonate than the parent cells. The fusion product showed priming activity for DNA polymerase on bleomycin-damaged DNA and acid-depurinated DNA. The deduced amino acid sequence of mouse APEX nuclease exhibits a significant homology to those of exonuclease III of E. coli and ExoA protein of Streptococcus pneumoniae and an intensive homology with that of bovine AP endonuclease 1.     

Primary structure of rat liver serine dehydratase deduced from the cDNA sequence

The nucleotide sequence of serine dehydratase mRNA of rat liver has been determined from a recombinant cDNA clone, previously cloned in this laboratory, and from a recombinant cDNA clone screened from a primer-extended cDNA library. The sequence of 1322 nucleotides includes the entire protein coding region and noncoding regions on the 3'- and 5'-sides. The deduced polypeptide consists of 327 amino acid residues with a calculated molecular mass of 34,462 Da. Comparison of the amino acid sequences of the serine dehydratase polypeptide with those of biosynthetic threonine dehydratase of yeast and biodegradative threonine dehydratase of E. coli revealed various extents of homology. A heptapeptide sequence, Gly-Ser-Phe-Lys-Ile-Arg-Gly, which is the pyridoxal-binding site in the yeast and E. coli threonine dehydratases was found as a highly conserved sequence.     

Cloning and nucleotide sequence of the aspartase gene of Escherichia coli W.

The aspA gene of Escherichia coli W which encodes aspartase was cloned into the plasmid vector pBR322. The nucleotide sequences of aspA and its flanking regions were determined. The aspA gene encodes a protein with a molecular weight of 52,224 consisted of 477 amino acid residues. The amino acid sequence of the protein predicted from the nucleotide sequence was consistent with those of the NH2- and COOH-terminal regions and also with the amino acid composition of the purified aspartase determined previously. Potential promoter and terminator sequences for aspA were also found in the determined sequence.     

Molecular cloning and primary structure of cDNA encoding the catalytic domain of rat liver aspartyl-tRNA synthetase

A cDNA clone encoding rat liver aspartyl-tRNA synthetase was isolated by probing a lambda gt11 recombinant cDNA expression library with antibodies directed against the corresponding polypeptide from sheep liver. The 1930-base pairs-long cDNA insert allowed the expression in Escherichia coli of an active enzyme of mammalian origin. The nucleotide sequence of that cDNA, corresponding to the DRS1 gene, was determined. The open reading frame of DRS1 corresponds to a protein of Mr = 57,061, in good agreement with the previously determined molecular weight of the purified enzyme. The deduced amino acid sequence shows extensive homologies with that of yeast cytoplasmic aspartyl-tRNA synthetase, more than 50% of the residues being identical. In rat liver, aspartyl-tRNA synthetase occurs in two distinct forms: a dimeric enzyme and a component of a multienzyme complex comprising the nine aminoacyl-tRNA synthetases specific for arginine, aspartic acid, glutamic acid, glutamine, isoleucine, leucine, lysine, methionine, and proline. The primary structure of the DRS1 gene product is discussed in relation to the occurrence of two distinct forms of that enzyme.     

Nucleotide sequence of a full-length cDNA coding for 3-methylcholanthrene-induced rat liver cytochrome P-450MC

We constructed a full-length cDNA coding for 3-methylcholanthrene-inducible rat liver cytochrome P-450MC by the method of Okayama and Berg. The isolated clone pAU157 contained the cDNA insert of 2.7 kb in length. Sequence analysis of the cDNA insert revealed that the amino acid sequence of cytochrome P-450MC was composed of 523 amino acid residues, including the initial 22 N-terminal amino acids whose sequence was determined with the purified protein. The primary structure was found to contain two highly conserved regions as pointed out from comparisons of the reported amino acid sequences of cytochrome P-450 species. The predicted molecular weight of the apoprotein was 59,300 daltons. Therefore, we concluded that the amino acid sequence determined here is for cytochrome P-450MC, probably corresponding to cytochrome P-450c.     

Isolation and sequencing of a cDNA clone encoding acid phosphatase in rat liver lysosomes

A full length cDNA for acid phosphatase in rat liver lysosomes was isolated and sequenced. The predicted amino acid sequence comprises 423 residues (48,332 Da). A putative signal peptide of 30 residues is followed by the NH2-terminal sequence of lysosomal acid phosphatase (45,096 Da). The deduced NH2-terminal 18-residue sequence is identical with that determined directly for acid phosphatases purified from the rat liver lysosomal membranes. The primary structure deduced for acid phosphatase contains 9 potential N-glycosylation sites and a hydrophobic region which could function as a transmembrane domain. It exhibits 89% and 67% sequence similarities in amino acids and nucleic acids, respectively, to human lysosomal acid phosphatase. The amino acid sequence of the putative transmembrane segment shows a complete similarity to that of the human enzyme. Northern blot hybridization analysis identified a single species of acid phosphatase mRNA (2.2 kbp in length) in rat liver.     

Rat cytosolic aspartate aminotransferase: molecular cloning of cDNA and expression in Escherichia coli

cDNA clones for rat cytosolic aspartate aminotransferase (cAspAT, L-aspartate:2-oxoglutarate aminotransferase) [EC 2.6.1.1] were isolated from a rat cDNA library, and the primary structure of the gene for cAspAT was deduced from its cDNA sequence. Rat cAspAT consists of 412 amino acids and its molecular weight is 46,295. The deduced amino acid sequence of rat cAspAT was compared with the sequences of AspATs from other species. The degree of sequence identities of rat/mouse cAspAT, rat/pig cAspAT, rat/chicken cAspAT, rat/pig mAspAT, and rat/Escherichia coli AspAT were 97.1, 89.6, 81.7, 48.1, and 41.2%, respectively. A coding region of rat cAspAT cDNA was inserted into E. coli expression vector pUC9, and enzymatically active cAspAT was expressed as a beta-galactosidase-cAspAT hybrid protein. This hybrid protein represented about 18% of the soluble proteins in E. coli and its kinetic properties were comparable with those of cAspAT preparations purified from rat liver.     

Purification, molecular cloning, and immunohistochemical localization of dipeptidyl peptidase II from the rat kidney and its identity with quiescent cell proline dipeptidase

We purified dipeptidyl peptidase II (DPP II) to homogeneity from rat kidney and determined its physicochemical properties, including its molecular weight, substrate specificity, and partial amino acid sequence. Furthermore, we screened a rat kidney cDNA library, isolated the DPP II cDNA and determined its structure. The cDNA was composed of 1,720 base pairs of nucleotides, and 500 amino acid residues were predicted from the coding region of cDNA. Human quiescent cell proline dipeptidase (QPP) cloned from T-cells is a 58-kDa glycoprotein existing as a homodimer formed with a leucine zipper motif. The levels of amino acid homology were 92.8% (rat DPP II vs. mouse QPP) and 78.9% (rat DPP II vs. human QPP), while those of nucleotide homology were 93.5% (rat DPP II vs. mouse QPP) and 79.4% (rat DPP II vs. human QPP). The predicted amino acid sequences of rat DPP II and human and mouse QPP possess eight cysteine residues and a leucine zipper motif at the same positions. The purified DPP II showed similar substrate specificity and optimal pH to those of QPP. Consequently, it was thought that DPP II is identical to QPP. Northern blot analysis with rat DPP II cDNA revealed prominent expression of DPP II mRNA in the kidney, and the order for expression was kidney > testis > or = heart > brain > or = lung > spleen > skeletal muscle > or = liver. In parallel with Northern blot analysis, the DPP II antigen was detected by immunohistochemical staining in the cytosol of epithelial cells in the kidney, testis, uterus, and cerebrum.     

苜蓿夜蛾中肠丝氨酸蛋白酶cDNA的克隆、序列分析及原核表达

【目的】本研究旨在从苜蓿夜蛾Heliothis viriplaca中肠克隆出丝氨酸蛋白酶（serine protease, SP）基因的cDNA序列,测定原核表达后的蛋白经纯化及复性后的活性。【方法】运用RT-PCR和cDNA末端快速扩增方法（rapid amplification of cDNA ends, RACE）克隆苜蓿夜蛾幼虫中肠丝氨酸蛋白酶cDNA全序列,用大肠杆菌Escherichia coli表达系统进行表达。重组蛋白经纯化后,利用梯度透析法进行复性,以BApNA为底物,进行活性测定。【结果】克隆获得的苜蓿夜蛾中肠丝氨酸蛋白酶基因命名为HvSP（GenBank登录号：JX866720）,该基因全长880 bp,开放阅读框长762 bp,编码254个氨基酸,推测分子量和pI值分别为26.9 kDa和9.49。由HvSP推导的氨基酸与鳞翅目昆虫SP氨基酸序列的一致性在52%~95%之间,其中与棉铃虫Helicoverpa armigera SP（GenBank登录号：CAA72962）的氨基酸序列一致性最高,达95%。成功构建重组载体pET21b-HvSP进行原核表达,Western-blot鉴定确定为目的蛋白。蛋白可溶性分析发现重组蛋白为包涵体。在Glycine-NaOH缓冲液中,当pH为10.0时,复性的重组蛋白活性达到最高,为35.74 U/mL。【结论】本研究在苜蓿夜蛾体内获得了一个新的丝氨酸蛋白酶基因,且原核表达后的重组蛋白经过变性、纯化及复性后具有活性。该结果为进一步研究丝氨酸蛋白酶在鳞翅目昆虫体内的生理功能奠定了基础。     

Purification and characterization of recombinant murine immune interferon

The recombinant murine immune interferon (rMu-IFN-gamma) was purified to homogeneity from Escherichia coli harboring the expression vector of murine IFN-gamma. The purified rMu-IFN-gamma showed an Mr of 15 000 in SDS-polyacrylamide gel electrophoresis. Results of amino acid analysis, amino- and carboxyl-terminal analyses and peptide mapping of rMu-IFN-gamma suggest that it has the complete protein sequence predicted on the basis of cDNA except for lack of four amino acid residues from the mature carboxyl-terminus.     

Primary structure of rat liver alkaline phosphatase deduced from its cDNA.

Rat liver alkaline phosphatase (ALP) was markedly induced by treatment of rats by bile-duct ligation and colchicine injection. Taking this advantage for enrichment of ALP mRNA, we constructed a lambda gt11 liver cDNA library using polyadenylated RNA prepared from the treated rat liver, and isolated an ALP cDNA clone. The 2165 bp cDNA contained an open reading frame that encodes a 524-amino-acid-residue polypeptide with a predicted molecular mass of 57737 Da. The precursor protein contained a presumed signal peptide of 17 amino acid residues followed by 28 amino acid residues identical with the N-terminal sequence determined from the purified rat liver ALP. It was also confirmed that amino acid sequences of two CNBr-cleavage peptides obtained from liver ALP were contained within the cDNA-encoded protein. Five possible N-linked glycosylation sites were found in the molecule and a highly hydrophobic amino acid sequence at the C-terminus. The deduced polypeptide of rat liver ALP showed 88% homology to that of the human liver-type enzyme in osteosarcoma cells. RNA blot hybridization analysis identified a single species of ALP mRNA with 2.7 kb in both the control and the treated rat livers. An approx. 20-fold increase of the mRNA was detected in the treated liver at 12 h after the onset of stimulation, compared with that in the control liver.