Overproduction from a cellulase gene with a high guanosine-plus-cytosine content in Escherichia coli.

A recombinant exoglucanase was expressed in Escherichia coli to a level that exceeded 20% of total cellular protein. To obtain this level of overproduction, the exoglucanase gene coding sequence was fused to a synthetic ribosome-binding site, an initiating ATG, and placed under the control of the leftward promoter of bacteriophage lambda contained on the runaway replication plasmid vector pCP3 (E. Remaut, H. Tsao, and W. Fiers, Gene 22:103-113, 1983). With the exception of an inserted asparagine adjacent to the initiating ATG, the highly expressed exoglucanase is identical to the native exoglucanase. The overproduced exoglucanase can be isolated easily in an enriched form as insoluble aggregates, and exoglucanase activity can be recovered by solubilization of the aggregates in 6 M urea or 5 M guanidine hydrochloride. Since the codon usage of the exoglucanase gene is so markedly different from that of E. coli genes, the overproduction of the exoglucanase in E. coli indicates that codon usage may not be a major barrier to heterospecific gene expression in this organism.     

Cloning and expression in Escherichia coli of a cellulase gene from Ruminococcus flavefaciens.

An endoglucanase gene of Ruminococcus flavefaciens FD1 was cloned on the vector pEcoR251 to form the recombinant plasmid pMEB200. The cloned endoglucanase gene showed carboxymethylcellulase enzyme activity but no degradation of Avicel (FMC Corp., Philadelphia, Pa.) or filter paper. Carboxymethylcellulase activity was found during the late-exponential-growth phase and accumulated in the periplasmic fraction. Enzyme production was not subject to catabolite repression by glucose.     

Overproduction of a selenocysteine-containing polypeptide in Escherichia coli: the fdhF gene product

The fdhF gene of Escherichia coli codes for the selenocysteine-including protein subunit of formate dehydrogenase H. The protein subunit consists of 715 amino acid residues containing a single selenocysteine residue at position 140 which is encoded by a UGA codon. The decoding of this opal termination codon occurs under anaerobic growth conditions by means of a specific tRNA, i.e. the selC gene product. The ability of E. coli cells to overproduce a selenopolypeptide was examined using the fdhF gene as a model system. Surprisingly, E. coli was able to synthesize the fdhF gene product at the level of approximately 12% of the total cellular protein. This was achieved by cloning fdhF in a multicopy plasmid together with a synthetic selC gene under the Ipp promoter. FdhF production was absolutely dependent upon the addition of selenium to the culture medium and was almost completely blocked in the presence of oxygen. The product was specifically labelled with 75Se, proving that it consisted of a selenoprotein. The product was purified to homogeneity and shown to exhibit the catalytic properties characteristic of formate dehydrogenase H.     

Overproduction of a stable acetaldehyde dehydrogenase with high affinity to acetaldehyde by Escherichia coli expressing the ACOD gene of Alcaligenes eutrophus

Summary The acoD gene of Alcaligenes eutrophus, which encodes a very stable NAD dependent aldehyde dehydrogenase with high affinity toward acetaldehyde (K _m = 4M), was overexpressed in Escherichia coli. Plasmid pDel087, a deletion derivative of a plasmid constructed recently (Priefert et al., 1992), conferred acetaldehyde dehydrogenase activity of 2.5 U/mg of protein to E. coli, which was about 8-fold higher than the activity in ethanol-grown cells of A. eutrophus.     

Overproduction of Escherichia coli NusA protein

The nusA gene of Escherichia coli has been cloned into the plasmid vector pKC30 under the control of the inducible lambda pL promoter. When a strain carrying this plasmid is induced, NusA protein is overproduced more than 100-fold and constitutes 20-30% of the total cellular protein. The NusA protein purified from this strain appears identical to authentic NusA protein in its migration on SDS polyacrylamide gels and on isoelectric focusing gels. It is also able to function properly in in vitro termination and antitermination assays and in its ability to bind to E. coli core RNA polymerase.     

Cloning of the cellulase gene from Ruminococcus albus and its expression in Escherichia coli

The gene for cellulase from Ruminococcus albus F-40 was cloned in Escherichia coli HB101 with pBR322. A 3.4-kilobase-pair HindIII fragment encoding cellulase hybridized with the chromosomal DNA of R. albus. The Ouchterlony double-fusion test gave a single precipitation line between the cloned enzyme and the cellulase from R. albus. The size of the cloned fragment was reduced by using HindIII and EcoRI. The resulting active fragment had a size of 1.9 kilobase pairs; and the restriction sites EcoRI, BamHI, PvuII, EcoRI, PvuII, and HindIII, in that order, were ligated into pUC19 at the EcoRI and HindIII sites (pURA1). Cellulase production by E. coli JM103(pURA1) in Luria-Bertani broth was remarkably enhanced, up to approximately 80 times, by controlling the pH at 6.5 and by reducing the concentration of NaCl in the broth to 80 mM.     

Cloning of the cellulase gene from Penicillium funiculosum and its expression in Escherichia coli

A gene of Penicillium funiculosum encoding an endoglucanase was cloned and expressed in Escherichia coli using the lacZ promoter of vector pUC 18. The gene product hydrolyzed carboxymethyl cellulose and showed strong cross reactivity with P. funiculosum anticellulases.     

A recombinant cellulolytic Escherichia coli: Cloning of the cellulase gene and characterization of a bifunctional cellulase

A genomic library of Bacillus subtilis CD4 was constructed in Escherichia coli JM83. A clone designated as E. coli pBcelR was identified which formed blue colony in presence of 5-bromo-4-chloro-3-indolyl--d-glucopyranoside (X-Glu) and hydrolysed carboxymethyl cellulose (CMC). The clone E. coli (pBcelR) expressed both cellobiase and endoglucanase activities and contained an insert of 1.2 kb. E. coli pBcelR encoded a protein of 12.9 kDa which was endowed with bifunctional (endoglucanase and cellobiase) activities. In recombinant E. coli, the encoded protein and enzyme activity were localized in periplasm. Recombinant E. coli pBcelR utilized CMC, cellobiose and soluble cellulose as sole carbon source.     

Overproduction of d-xylose isomerase in Escherichia coli by cloning the d-xylose isomerase gene

The Escherichia coli d-xylose isomerase (d-xylose ketol-isomerase, EC 5.3.1.5) gene, xylA, has been cloned on various E. coli plasmids. However, it has been found that high levels of overproduction of the d-xylose isomerase, the protein product of the xylA gene, cannot be accomplished by cloning the intact gene on high copy-number plasmids alone. This is believed to be due to the fact that the expression of the gene through its natural promoter is highly regulated in E. coli. In order to overcome this, the xylA structural gene has been fused with other strong promoters such as tac and lac, resulting in the construction of a number of fused genes. Analysis of the E. coli transformants containing the fused genes, cloned on high copy-number plasmids, indicated that a 20-fold overproduction of the enzyme can now be obtained. It is expected that overproduction of the enzyme in E. coli can still be substantially improved through additional manipulation with recombinant DNA techniques.     

Cloning of the cellulase gene from Ruminococcus albus and its expression in Escherichia coli.

The gene for cellulase from Ruminococcus albus F-40 was cloned in Escherichia coli HB101 with pBR322. A 3.4-kilobase-pair HindIII fragment encoding cellulase hybridized with the chromosomal DNA of R. albus. The Ouchterlony double-fusion test gave a single precipitation line between the cloned enzyme and the cellulase from R. albus. The size of the cloned fragment was reduced by using HindIII and EcoRI. The resulting active fragment had a size of 1.9 kilobase pairs; and the restriction sites EcoRI, BamHI, PvuII, EcoRI, PvuII, and HindIII, in that order, were ligated into pUC19 at the EcoRI and HindIII sites (pURA1). Cellulase production by E. coli JM103(pURA1) in Luria-Bertani broth was remarkably enhanced, up to approximately 80 times, by controlling the pH at 6.5 and by reducing the concentration of NaCl in the broth to 80 mM.     

外源基因在大肠杆菌中的高效表达

为了提高外源蛋白在大杨杆菌中的表达量,人们对大肠杆菌表达系统进行了许多研究。作者综述了有关外源基因在大肠杆菌中高效表达的研究进展。     

Overproduction and crystallization of tryptophanase from recombinant cells of Escherichia coli

We have cloned the tryptophanase structural gene from Escherichia coli B/1t7-A into E. coli K-12 MD55 with a vector plasmid, pBR322. The cloned cells produced a large amount of the enzyme corresponding to more than 30% of the total soluble protein. With the enzyme obtained by this overproduction system, we have prepared three different crystals of tryptophanase, apo-enzyme, holo-enzyme, and a complex of holo-enzyme and L-alanine, by using polyethylene glycol 4000 or potassium phosphate as a precipitant and the hanging drop method. These single crystals appeared to be suitable for X-ray diffraction analysis.     

Overproduction and preliminary crystallographic study of ribonuclease H from Escherichia coli

To facilitate the preparation of ribonuclease H from Escherichia coli in an amount sufficient for crystallographic studies, we have constructed an overproduction system for the enzyme. The structural gene for the enzyme was subcloned from pSK750 (Kanaya, S., and Crouch, R. J. (1983) J. Biol. Chem. 258, 1276-1281) to make a plasmid vector pPL801, in which the gene was under the control of bacteriophage lambda PL promoter. Thermal induction of the gene accumulated the enzyme in E. coli N4830-1 to approximately 8% of the total cytosolic protein. The level of production of the enzyme in N4830-1 harboring pPL801 was 14 mg/liter culture, which was 3000 times as high as that in the host cell. The enzyme was purified with a yield of more than 80% and crystallized by utilizing the property that the solubility of the enzyme decreased at pH values close to its isoelectric point (pI = 9). Crystals were grown by successive seeding (hanging drop method) for x-ray crystallographic analysis. The crystals belong to space group P212121 with unit cell dimensions of alpha = 44.1 A, b = 87.0 A, c = 35.5 A and contain one molecule in an asymmetric unit. They diffracted x-rays beyond 2.5 A resolution.     

Overproduction and preliminary X-ray characterization of aspartate aminotransferase from Escherichia coli

The aspartate aminotransferase of Escherichia coli was overproduced in cells after genetic manipulation, and was crystallized from a polyethylene glycol solution, pH 7.0. The crystals obtained were of good quality and had diffractions extending beyond 2.4 A. The space group and unit cell dimensions were determined with a precession camera and a four-circle diffractometer to be C222(1), and a = 157.1 A, b = 85.5 A, and c = 79.7 A, respectively. Only one protein subunit is contained in an asymmetric unit.     

Overproduction and large-scale preparation of deoxyuridine triphosphate nucleotidohydrolase from Escherichia coli

A recombinant plasmid, pHW1, directing the overproduction of the enzyme deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase, EC 3.6.1.23) from Escherichia coli has been constructed. A 1900-base DNA fragment carrying the structural gene for the enzyme (dut) has been recloned into a runaway replication vector that also carries the strong leftward promoter (pL) of bacteriophage lambda. Upon temperature shift, an E. coli strain carrying the new plasmid gives an increase in dUTPase activity of about 600-fold in rich medium compared to wild-type bacteria. The 64-kDa protein corresponding to the mature form of the enzyme reaches 20% of the total protein content of the bacterial cell. Using this strain, a simplified procedure has been developed for the purification of dUTPase. The purification steps consist of extraction of the cytoplasmic proteins, ammonium sulfate precipitation, anion-exchange chromatography and gel filtration on FPLC. The new overproducing plasmid and the simplified purification procedure developed will make it possible to purify dUTPase in sufficient amounts for detailed characterization studies.     

Overproduction of transcription termination factor Rho in Escherichia coli

A plasmid system has been constructed which allows high-level expression of the rho gene of Escherichia coli under the control of the pL promoter and the N-antitermination regulatory system of bacteriophage lambda. The pL-directed synthesis of Rho crucially depends on the lambda N gene product and is promoted most effectively when this product is supplied from the N gene cloned on a separate compatible plasmid with a moderate copy number. The requirement for N can be circumvented partly, but not completely, by deletion of the region preceding the rho structural gene. Attempts were also made to optimize the construction of rho-expression plasmids by adjusting the orientation and location of pL and rho inserts on the pBR322 vector. With optimal conditions, Rho protein is overexpressed 100-fold and can become as much as 10% of the total cellular protein. Using this plasmid system, Rho can be purified with a yield of more than 20 mg from 10 g of induced cells.     

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.     

Overproduction of d-hydantoinase and carbamoylase in a soluble form in Escherichia coli

The production of d-hydantoinase and carbamoylase from Agrobacterium radiobacter NRRL B11291 using T7 and trc promoters, respectively, was found to cause protein aggregates in Escherichia coli. We initiated a systematic study aimed at overproducting these two proteins in a soluble form. As a result, the protein aggregate from carbamoylase overproduction could be alleviated with the aid of GroEL/GroES. In contrast, the production of a high level of d-hydantoinase in an active form can be achieved at low temperature (25 °C) or by the coproduction of DnaJ/DnaK. Overall, with such approaches both recombinant proteins gain more than a four-fold increase in enzyme activity. In addition, by fusion with thioredoxin, d-hydantoinase activity can be increased 25% more than the unfused counterpart in the presence of DnaJ/DnaK. These results indicate the success of our approaches to overproducing d-hydantoinase and carbamoylase in a soluble form in E. coli. Received: 26 November 1999 / Received revision: 28 February 2000 / Accepted: 10 March 2000     

Overproduction in Escherichia coli and Characterization of a Soybean Ferric Leghemoglobin Reductase

We previously cloned and sequenced a cDNA encoding soybean ferric leghemoglobin reductase (FLbR), an enzyme postulated to play an important role in maintaining leghemoglobin in a functional ferrous state in nitrogen-fixing root nodules. This cDNA was sub-cloned into an expression plasmid, pTrcHis C, and overexpressed in Escherichia coli. The recombinant FLbR protein, which was purified by two steps of column chromatography, was catalytically active and fully functional. The recombinant FLbR cross-reacted with antisera raised against native FLbR purified from soybean root nodules. The recombinant FLbR, the native FLbR purified from soybean (Glycine max L.) root nodules, and dihydrolipoamide dehydrogenases from pig heart and yeast had similar but not identical ultraviolet-visible absorption and fluorescence spectra, cofactor binding, and kinetic properties. FLbR shared common structural features in the active site and prosthetic group binding sites with other pyridine nucleotide-disulfide oxidoreductases such as dihydrolipoamide dehydrogenases, but displayed different microenvironments for the prosthetic groups.