Molecular characterization of potato fumarate hydratase and functional expression in Escherichia coli.

The tricarboxylic acid cycle enzyme fumarase (fumarate hydratase; EC 4.2.1.2) catalyzes the reversible hydration of fumarate to L-malate. We report the molecular cloning of a cDNA (StFum-1) that encodes fumarase from potato (Solanum tuberosum L.). RNA blot analysis demonstrated that StFum-1 is most strongly expressed in flowers, immature leaves, and tubers. The deduced protein contains a typical mitochondrial targeting peptide and has a calculated molecular mass of 50.1 kD (processed form). Potato fumarase complemented a fumarase-deficient Escherichia coli mutation for growth on minimal medium that contains acetate or fumarate as the sole carbon source, indicating that functional plant protein was produced in the bacterium. Antiserum raised against the recombinant plant enzyme recognized a 50-kD protein in wild-type but not in StFum-1 antisense plants, indicating specificity of the immunoreaction. A protein of identical size was also detected in isolated potato tuber mitochondria. Although elevated activity of fumarase was previously reported for guard cells (as compared with mesophyll cells), additional screening and genomic hybridization data reported here do not support the hypothesis that a second fumarase gene is expressed in potato guard cells.     

Molecular cloning and functional expression of d-arabitol dehydrogenase gene from Gluconobacter oxydans in Escherichia coli

A NADP-dependent d-arabitol dehydrogenase gene was cloned from Gluconobacter oxydans CGMCC 1.110 and functionally expressed in Escherichia coli. With d-arabitol as sole carbon source, E. coli transformants grew rapidly in minimal medium, and produced d-xylulose. The enzymatic properties of the 29kDa enzyme were documented. The DNA sequence surrounding the gene suggested that it is part of an operon with several components of a sugar alcohol transporter system, and the d-arabitol dehydrogenase gene belongs to the short-chain dehydrogenase family.     

Purification and characterization of catalytically active precursor of rat liver mitochondrial aldehyde dehydrogenase expressed in Escherichia coli

The cDNA coding for the precursor (p-ALDH) or mature (m-ALDH) rat liver mitochondrial aldehyde dehydrogenase was cloned in an expression vector pT7-7 and expressed in Escherichia coli strain BL21 (DE3)/plysS. The p-ALDH expressed in E. coli was a soluble tetrameric protein. It exhibited virtually the same specific activity and KmS for substrates as m-ALDH. N-terminal sequencing of isolated p-ALDH provided the evidence that the catalytic activity was not derived from a partially processed mature-like enzyme. The assembly states of both p-ALDH and m-ALDH synthesized in a rabbit reticulocyte lysate were also determined. Both of them were monomers and could not bind to a 5'-AMP-Sepharose column, showing that the monomeric form of the enzyme is inactive. The stabilities in vivo and in vitro were compared between p-ALDH and m-ALDH expressed in E. coli. p-ALDH was less stable than was m-ALDH both in vivo and in vitro. Thus, although the conformations of p-ALDH and m-ALDH are similar, the presence of signal peptide is a destabilizing factor to the p-ALDH. p-ALDH expressed in E. coli could bind to and be translocated into rat liver mitochondria, however, with lower efficiency when compared to the import of p-ALDH synthesized in reticulocyte lysate.     

Cloning and expression of the Clostridium thermocellum L-lactate dehydrogenase gene in Escherichia coli and enzyme characterization

The structural gene for L-lactate dehydrogenase (LDH) (EC.1.1.1.27) from Clostridium thermocellum 27405 was cloned in Escherichia coli by screening the Lambda Zap II phage library of C. thermocellum genomic DNA. In one positive clone, an open reading frame of 948 base pairs corresponded to C. thermocellum ldh gene encoding for the predicted 315-residue protein. The ldh gene was successfully expressed in E. coli FMJ39 (ldh mutant) under the lac promoter. The recombinant enzyme was partially purified from E. coli cell extracts and its kinetic properties were determined. Clostridium thermocellum LDH was shown to catalyze a highly reversible reaction and to be an allosteric enzyme that is activated by fructose-1,6-diphosphate (FDP). For pyruvate, partially purified LDH had Km and Vmax values of 7.3 mmol/L and 87 micromol/min, respectively, and in the presence of FDP, a 24-fold decrease in Km and a 5.7-fold increase in Vmax were recorded. The enzyme exhibited no marked catalytic activity for lactate in the absence of FDP, whereas Km and Vmax values were 59.5 mmol/L and 52 micromol/min, respectively, in its presence. The enzyme did not lose activity when incubated at 65 degrees C for 5 min.     

Molecular cloning of eel growth hormone cDNA and its expression in Escherichia coli

cDNA clones coding for growth hormone (eGH) of Japanese eel (Anguilla japonica) have been isolated from a cDNA library prepared from pituitary gland poly(A)+ RNA. The nucleotide sequence of the eGH cDNA was determined. It codes for the prehormone of 209 amino acids (aa) including a putative signal peptide of 19 aa. The deduced amino acid sequence was identical with that determined for eGH protein. The primary structure of eGH was compared with those of other species growth hormones (chum salmon, chicken, rat, and human). Mature eGH was expressed in Escherichia coli harboring a plasmid in which the eGH cDNA was under control of the phage lambda pL promoter. Recombinant eGH polypeptide was immunoreactive to rabbit antiserum against natural eGH. Furthermore, eGH derivative with amino-terminal deletion (delta 1-3 eGH) was produced in E. coli reaching up to 5% of total cellular proteins.     

大鼠OB基因克隆及其在大肠杆菌中的表达

采用ＲＴＰＣＲ技术扩增大鼠ＯＢｃＤＮＡ编码区序列。ＰＣＲ产物酶切后定向克隆至ｐＵＣ１９质粒。经核苷酸序列测定表明与文献报道的大鼠ＯＢｃＤＮＡ编码区序列一致。继之构建了ｐＢＶ２２０ｒＯＢ表达质粒并获得了ＯＢ基因在大肠杆菌中的特异表达。大鼠ＯＢ基因产物的获得为研究肥胖与某些非传染性疾病（如糖尿病、高血压病）间的关系提供了条件     

Molecular characterization of Escherichia coli FtsE and FtsX

The genes ftsE and ftsX are organized in one operon together with ftsY. FtsY codes for the receptor of the signal recognition particle (SRP) that functions in targeting a subset of inner membrane proteins. We have found no indications for a structural relationship between FtsE/X and FtsY. Evidence is presented that FtsE and FtsX form a complex in the inner membrane that bears the characteristics of an ATP-binding cassette (ABC)-type transporter. FtsE is a hydrophilic nucleotide-binding protein that has a tendency to dimerize and associates with the inner membrane through an interaction with the integral membrane protein FtsX. An FtsE null mutant showed filamentous growth and appeared viable on high salt medium only, indicating a role for FtsE in cell division and/or salt transport.     

Coupled expression of MhpE aldolase and MhpF dehydrogenase in Escherichia coli

MhpE (4-hydroxy-2-ketovalerate aldolase) and MhpF [acetaldehyde dehydrogenase (acylating)] are responsible for the last two reactions in the 3-(3-hydroxyphenyl)propionate (3-HPP) catabolic pathway in Escherichia coli, which is homologous to the meta-cleavage pathway in Pseudomonas species. Here, we report that the MhpE aldolase is associated with the MhpF dehydrogenase and that MhpF is indispensable for the folding of MhpE. Moreover, our results suggest that the mhpF and mhpE genes are translationally coupled through a reinitiation mechanism. This reinitiation mechanism may function in ensuring that the expression of mhpE occurs only when MhpF is available for the formation of a complex.     

Molecular cloning of giant panda pituitary prolactin cDNA and its expression in Escherichia coli

cDNA encoding pituitary (PRL) of giant panda was obtained using RT-PCR and expressed in E. coli. The results revealed that panda PRL cDNA encodes a precursor protein of 229 amino acids including a putative signal peptide of 30 amino acids and a mature protein of 199 residues with one potential N-glycosylation site. Sequence comparison indicated that panda PRL shares a high degree of identity to other known PRL sequences ranging from 98% with mink PRL to about 50% with rodent PRL. Six cysteine residues and 29 conserved residues distributed in four domains (PD1, PD2, PD3, and PD4) of PRL were observed. through multiple sequence alignment. Fourteen key residues of binding sites 1 and 2 involved in receptor binding are conserved in panda PRL. GST fused recombinant panda PRL protein was efficiently expressed with the form of insoluble inclusion bodies in E. coli BL21 transformed with a pGEX-4T-1 expression vector containing the DNA sequence encoding mature panda PRL. Western blot analysis indicated that GST-panda PRL recombinant protein could be recognized by antibody against human PRL. Our results would contribute to further elucidating the structural and functional characteristics of pituitary PRL and provide a basis for the production of recombinant panda prolactin for future use in the breeding of giant panda.     

Molecular characterization of the beta-N-acetylglucosaminidase of Escherichia coli and its role in cell wall recycling

The beta-N-acetylglucosaminidase of Escherichia coli was found to have a novel specificity and to be encoded by a gene (nagZ) that maps at 25.1 min. It corresponds to an open reading frame, ycfO, whose predicted amino acid sequence is 57% identical to that of Vibrio furnissii ExoII. NagZ hydrolyzes the beta-1,4 glycosidic bond between N-acetylglucosamine and anhydro-N-acetylmuramic acid in cell wall degradation products following their importation into the cell during the process for recycling cell wall muropeptides. From amino acid sequence comparisons, the novel beta-N-acetylglucosaminidase appears to be conserved in all 12 gram-negative bacteria whose complete or partial genome sequence data are available.     

Molecular characterization of Escherichia coli NAD kinase.

NAD kinase was purified to homogeneity from Escherichia coli MG1655. The enzyme was a hexamer consisting of 30 kDa subunits and utilized ATP or other nucleoside triphosphates as phosphoryl donors for the phosphorylation of NAD, most efficiently at pH 7.5 and 60 degrees C. The enzyme could not use inorganic polyphosphates as phosphoryl donors and was designated as ATP-NAD kinase. The N-terminal amino-acid sequence of the purified enzyme was encoded by yfjB, which had been deposited as a gene of unknown function in the E. coli whole genomic DNA sequence database. yfjB was cloned and expressed in E. coli BL21(DE3)pLysS. The purified product (YfjB) showed NAD kinase activity, and was identical to ATP-NAD kinase purified from E. coli MG1655 in molecular structure and other enzymatic properties. The deduced amino-acid sequence of YfjB exhibited homology with that of Mycobacterium tuberculosis inorganic polyphosphate/ATP-NAD kinase [Kawai, S., Mori, S., Mukai, T., Suzuki, S., Hashimoto, W., Takeshi, Y. & Murata, K. (2000) Biochem. Biophys. Res. Commun. 276, 57-63], and those of many hypothetical proteins for which functions have not yet been revealed. The YfjB homologues were considered to be NAD kinases and alignment of their sequences revealed highly conserved regions, XXX-XGGDG-XL and DGXXX-TPTGSTAY, where X represents a hydrophobic amino-acid residue.     

Purification and characterization of osmoregulatory betaine aldehyde dehydrogenase of Escherichia coli

The osmoregulatory NAD-dependent betaine aldehyde dehydrogenase (betaine aldehyde:NAD oxidoreductase, EC 1.2.1.8), of Escherichia coli, was purified to apparent homogeneity from an over-producing strain carrying the structural gene for the enzyme (betB) on the plasmid vector pBR322. Purification was achieved by ammonium sulfate fractionation of disrupted cells, followed by affinity chromatography on 5'-AMP Sepharose, gel-filtration and ion-exchange chromatography. The amino acid composition was determined. The dehydrogenase was found to be a tetramer with identical 55 kDa subunits. Both NAD and NADP could be used as cofactor for the dehydrogenase, but NAD was preferred. The dehydrogenase was highly specific for betaine aldehyde. None of the analogs tested functioned as a substrate, but several inhibited the enzyme competitively. The enzyme was not activated by salts at concentrations encountered during osmotic upshock, but it was salt tolerant, retaining 50% of maximal activity at 1.2 M K+. It is inferred that salt tolerance is an essential property for an enzyme participating in the cellular synthesis of an osmoprotectant.     

Overproduction of staphylokinase in Escherichia coli and its characterization

A recombinant plasmid which directs the overproduction in Escherichia coli of staphylokinase from Staphylococcus aureus has been constructed by placing the staphylokinase gene, sak, under the control of bacteriophage lambda PR promoter in the plasmid. When an E. coli strain having the plasmid was induced, the staphylokinase activity in the periplasmic fraction increased about 60-fold and the 15.5-kDa protein corresponding to the mature form reached about 25% of the periplasmic proteins. At the same time the 18.5-kDa protein corresponding to the precursor form was accumulated in the membrane fraction, showing that the processing and translocation of the sak gene product were restricted during high level of its synthesis. By using this strain, the mature staphylokinase has been easily purified to near homogeneity. The purification steps consisted of extraction of the periplasmic proteins by osmotic shock and CM-cellulose column chromatography. Two species of staphylokinase were identified after CM-cellulose column chromatography. Although their isoelectric points and NH2-terminal amino acid sequences were different, their specific activities were almost equal. These results strongly suggest that the NH2-terminal portion of staphylokinase is not important for its activity.     

Isolation and characterization of two types of cDNA for mitochondrial adenylate kinase and their expression in Escherichia coli

Two cDNA clones for mitochondrial adenylate kinase were isolated from a cDNA library of bovine liver poly(A)+ RNA by using synthetic oligodeoxynucleotides as probes. The clone containing a 0.9-kilobase insert had the reading frame for a 241-residue protein (AK2A), while the other clone containing a 1.6-kilobase insert had the frame for a 234-residue protein (AK2B). Nucleotide sequences of these two clones were the same in the 5' portion up to the coding sequence for the 233rd residue, but different in the remaining 3' portions. The reported amino acid sequence of mitochondrial adenylate kinase from bovine heart corresponded to AK2A. Neither AK2A nor AK2B had a cleavable NH2-terminal presequence as that found in other imported mitochondrial proteins. RNA blot analysis of poly(A)+ RNAs from bovine liver and heart revealed three species of mRNA with approximate sizes of 0.9, 1.4, and 1.7 kilobases. The 1.7- and 1.4-kilobase species were specific for AK2B, whereas the 0.9-kilobase species was specific for AK2A. In the liver, the 1.7-kilobase mRNA was more abundant, whereas in the heart the 0.9-kilobase mRNA was predominant. The 1.4-kilobase mRNA was present only in the heart. The AK2A- and AK2B-coding sequences were expressed in Escherichia coli cells under the control of trc promoter. Both the products reverted the temperature-sensitive phenotype of the adenylate kinase mutant of E. coli.     

Expression and characterization of human mitochondrial ferredoxin reductase in Escherichia coli.

Ferredoxin reductase (Fd-reductase) supplies reducing equivalents obtained from NADPH to mitochondrial cytochrome P450 enzymes via the small iron-sulfur protein ferredoxin. Two cDNAs (differing by the presence or absence of an 18-bp insert in the coding region) for the human Fd-reductase were subcloned into a newly constructed general purpose expression vector, p delta blue; protein expression under control of the bacteriophage lambda pL promoter was then induced in Escherichia coli. Western blot analysis of subcellular fractions indicated that Fd-reductase protein expressed from both plasmids was present in both inclusion bodies and soluble fractions. However, only the form lacking the insert exhibited Fd-reductase activity. The active material was purified and was found to have electrophoretic, chromatographic, optical, and circular dichroism properties comparable to the bovine homologue. The apparent Km of the expressed protein for NADPH was determined to be 0.7 +/- 0.1 microM and the apparent Km for human ferredoxin was found to be 106 +/- 8 nM. While yields of active enzyme were relatively low (approximately 0.1 mg/liter of culture), the production of Fd-reductase in E. coli will allow structural and mechanistic studies of the enzyme and its interactions with ferredoxin.