Thermostable tryptophan synthase ofBacillus stearothermophilus expressed inEscherichia coli

Summary The tryptophan synthase genes,trpA andtrpB, from a moderate thermophile,Bacillus stearothermophilus IFO13737, were expressed efficiently inEscherichia coli. The recombinant tryptophan synthase amounted to 22% of the soluble cellular protein, and was purified to homogeneity by three steps. The enzyme is more thermostable thanE.coli tryptophan synthase, especially the subunit. The enzyme is also more resistant to sodium dodecylsulfate and methanol thanE.coli enzyme.     

Stability of hyperproduction of D-serine deaminase and tryptophanase inEscherichia coli

Summary D-Serine deaminase hyperproducing mutants ofE. coli lose about one half of their original activity during passaging. On the other hand, the mutant hyperproducing tryptophanase is completely stable.     

A pH-regulated promoter for the expression of recombinant proteins inEscherichia coli

Summary A series of plasmid vectors have been developed which allows control of recombinant gene expression by altering the pH of the growth medium. Expression is controlled by the regulatory region of thecadA gene ofE. coli. Experiments using -galactosidase as the expressed gene have resulted in an induced expression of up to 60-fold when the pH of the growth medium is lowered from pH 7 to 5.5. Expression can also be induced by switching from aerobic to anaerobic growth environment. The pH and anaerobic effects are additive boosting the expression level of -galactosidase to a dramatic value of 200 fold. Finally, the pH-induction effect is fully reversible, a unique property which allows continuous control of gene expression using exiting pH monitoring and control equipment.     

Modulation of gene expression by the product offitA gene inEscherichia coli

Physiological parameters such as viability, gross RNA synthesis,β-galactosidase induction, development of phages T4, T7 andλ have been studied in temperature-sensitiveEscherichia coli strains harbouring fit A76,fit A24 andfit A76fit A24 mutations in rpoB⁺ andrpoB240 genetic backgrounds. The efficiently of expression of these functions is influenced by thefit A alleles depending upon the medium of growth and/or temperature. Strains harbouring therpoB240 mutation and thefit A76 mutation, either alone or together with thefit A24 mutation, are rifampicin-sensitive even at the perfssive temperature. The results suggest possible interaction between thefit A gene product and RNA polymerase invivo. This paper is dedicated to Proof. S. Krishnaswamy on his Sixty First Birthday.     

Cloning and expression of theClostridium thermocellum celS gene inEscherichia coli

Clostridium thermocellum ATCC 27405 produces an extremely complicated multi-component cellulase aggregate (cellulosome) highly active on crystalline cellulose. From the cellulosome, two subunits, CelS (or S _s ;M _r = 82 000) and CelL (or S _l , CipA;M _r = 250 000), have been identified as essential for crystalline cellulose degradation [Wu et al. (1988) Biochemistry 27:1703]. We have determined the DNA sequence of thecelS gene from four cloned DNA fragments encompassing this gene [Wang et al. (1993) J Bacteriol 175:1293]. To express the entirecelS gene inEscherichia coli, thecelS structural gene was amplified by the polymerase chain reaction (PCR) employing the PCR primers corresponding to sequences flanking the desired gene. This PCR product (2.1 x 10³ bases; 2.1 kb) was cloned into anE. coli expression vector pRSET B. Subsequent expression of the cloned gene resulted in a fusion protein (rCelS;M _r = 86 000) as inclusion bodies. The rCelS protein was recognized specifically by an anti-CelS antiserum in a Western blot analysis. The inclusion bodies were purified and solubilized in 5m urea. The refolded rCelS produced very little reducing sugar from carboxymethylcellulose. However, it showed a higher activity on the crystalline cellulose (Avicel) and an even higher activity on phosphoricacid-swollen Avicel. These results indicate that the CelS is an exoglucanase.     

Cloning and expression inEscherichia coli of arecA-like gene fromZymomonas mobilis

Intergeneric complementation ofEscherichia coli recA mutants was used to identify recombinant plasmids, within a genomic library derived fromZymomonas mobilis, that carryZ. mobilis recA-like gene. Screening of 1100 individualE. coli strains revealed four clones expressing therecA⁺ character. On restriction analysis, all four recombinant plasmids were found to be related and to exhibit a common 6.7-kb fragment. Consequently, one of the four recombinant plasmids, pZR27, was selected for further characterization. When introduced intoE. coli recA mutants, pZR27 restored resistance to methyl methane sulfonate, mitomycin-C, and UV irradiation, as well as recombination proficiency when measured by standard Hfr-mediated conjugation. The clonedrecA-like gene also restored the spontaneous and mitomycin-C-induced phage production. The origin of the insert in pZR27 from the chromosome ofZ. mobilis was confirmed by Southern transfer and DNA hybridization. However, no homology was found between therecA ofE. coli andZ. mobilis chromosomal insert DNA. TheZ. mobilis recA-like gene also encoded a major polypeptide of 38-kDa on SDS-PAGE.     

Reactions of O-acyl-L-serines with tryptophanase, tyrosine phenol-lyase, and tryptophan synthase

The reactions of tryptophanase, tyrosine phenol-lyase, and tryptophan synthase with a new class of substrates, the O-acyl-L-serines, have been examined. A method for preparation of O-benzoyl-L-serine in high yield from tert.-butyloxycarbonyl (tBoc)-L-serine has been developed. Reaction of the cesium salt of tBoc-L-serine with benzyl bromide in dimethylformamide gives tBoc-L-serine benzyl ester in excellent yield. Acylation with benzoyl chloride and triethylamine in acetonitrile followed by hydrogenolysis with 10% palladium on carbon in trifluoroacetic acid gives O-benzoyl-L-serine, isolated as the hydrochloride salt. O-Benzoyl-L-serine is a good substrate for beta-elimination or beta-substitution reactions catalyzed by both tryptophanase and tyrosine phenol-lyase, with Vmax values 5- to 6-fold those of the physiological substrates and comparable to that of S-(o-nitrophenyl)-L-cysteine. Unexpectedly, O-acetyl-L-serine is a very poor substrate for these enzymes, with Vmax values about 5% of those of the physiological substrates. Both O-acyl-L-serines are poor substrates for tryptophan synthase, measured either by the synthesis of 5-fluoro-L-tryptophan from 5-fluoroindole and L-serine catalyzed by the intact alpha 2 beta 2 subunit or by the beta-elimination reaction catalyzed by the isolated beta 2 subunit. With all three enzymes, the elimination of benzoate appears to be irreversible. These results suggest that the binding energy from the aromatic ring of O-benzoyl-L-serine is used to lower the transition-state barrier for the elimination reactions catalyzed by tryptophanase and tyrosine phenol-lyase. Our findings support the suggestion (M. N. Kazarinoff and E. E. Snell (1980) J. Biol. Chem. 255, 6228-6233) that tryptophanase undergoes a conformational change during catalysis and suggest that tyrosine phenol-lyase also may undergo a conformational change during catalysis.     

Regulation of tryptophan synthase gene expression in Chlamydia trachomatis

We previously reported that Chlamydia trachomatis expresses the genes encoding tryptophan synthase (trpA and trpB). The results presented here indicate that C. trachomatis also expresses the tryptophan repressor gene (trpR). The complement of genes regulated by tryptophan levels in C. trachomatis is limited to trpBA and trpR. trp gene expression was repressed if chlamydiae-infected HeLa cells were cultured the presence of tryptophan and induced if grown in tryptophan-depleted medium or in the presence of IFN-gamma. Furthermore, expression of the trp genes in strains which encode a functional tryptophan synthase is repressed when infected cells are cultured in the presence of the tryptophan precursor indole. Results from experiments with cycloheximide, an inhibitor of eukaryotic protein synthesis, indicate that in addition to the absolute size of the intracellular tryptophan pool, host competition for available tryptophan plays a key role in regulating expression of the trp genes. The tryptophan analogue, 5-fluorotryptophan, repressed trp gene expression and induced the formation of aberrant organisms of C. trachomatis. The growth-inhibitory properties of 5-fluorotryptophan could be reversed with exogenous tryptophan but not indole. In total, our results indicate that the ability to regulate trp gene expression in response to tryptophan availability is advantageous for the intracellular survival of this organism. Furthermore, the fact that C. trachomatis has retained the capacity to respond to tryptophan limitation supports the view that the in vivo antichlamydial effect of IFN-gamma is via the induction of the tryptophan-degrading enzyme, indoleamine 2,3-dioxygenase.     

Beta-elimination of indole from L-tryptophan catalyzed by bacterial tryptophan synthase: a comparison between reactions catalyzed by tryptophanase and tryptophan synthase

Although tryptophan synthase catalyzes a number of pyridoxal phosphate dependent beta-elimination and beta-replacement reactions that are also catalyzed by tryptophanase, a principal and puzzling difference between the two enzymes lies in the apparent inability of tryptophan synthase to catalyze beta-elimination of indole from L-tryptophan. We now demonstrate for the first time that the beta 2 subunit and the alpha 2 beta 2 complex of tryptophan synthase from Escherichia coli and from Salmonella typhimurium do catalyze a slow beta-elimination reaction with L-tryptophan to produce indole, pyruvate, and ammonia. The rate of the reaction is about 10-fold higher in the presence of the alpha subunit. The rate of indole production is increased about 4-fold when the aminoacrylate produced is converted to S-(hydroxyethyl)-L-cysteine by a coupled beta-replacement reaction with beta-mercaptoethanol. The rate of L-tryptophan cleavage is also increased when the indole produced is removed by extraction with toluene or by condensation with D-glyceraldehyde 3-phosphate to form indole-3-glycerol phosphate in a reaction catalyzed by the alpha subunit of tryptophan synthase. The amount of L-tryptophan cleavage is greatest in the presence of both beta-mercaptoethanol and D-glyceraldehyde 3-phosphate, which cause the removal of both products of cleavage. The cleavage reaction is not due to contaminating tryptophanase since the activity is not inhibited by (3R)-2,3-dihydro-L-tryptophan, a specific inhibitor of tryptophanase, but is inhibited by (3S)-2,3-dihydro-L-tryptophan, a specific inhibitor of tryptophan synthase. The cleavage reaction is also inhibited by D-tryptophan, the product of a slow racemization reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning and expression inEscherichia coli of entomotoxic protein gene fromBacillus thuringiensis subspecieskurstaki

A plasmid borne larvicidal crystal protein gene from B.thuringiensis subspecieskurstaki was cloned inEscherichia coli using a specific 20-mer oligonucleotide probe. The gene expressed inE. coli at a high level. TransgenicE. coli cells produced large irregular bodies which looked bright under phase contrast microscopy. The phase bright bodies released by sonic disruption of cells could be pelleted by centrifugation. Toxicity trials on the larvae ofSpodoptera litura showed that the pellet was antifeedant and toxic to the larvae. The supernatant was only mildly antifeedant. Even short term feeding of larvae on the toxin delayed the onset of pupation.     

Secretion expression of recombinant glucagon inEscherichia coli

A novel approach for the preparation of recombinant human glucagon was described. An expression vector pAGluT, containing phoA promoter, phoA signal peptide and glucagon gene, was constructed by means of genetic engineering.Escherichia coli strain YK537 was transformed with pAGluT. High-level secretory expression of recombinant human glucagon was achieved. The expression yield of recombinant human glucagon was found to be 80 mg/L, approximately 30% of the total proteins in supernatant. The biological activities and the physicochemical properties of the purified recombinant human glucagon were found to be the same as that of native glucagon. In addition, our results suggested that phoA expression system may be suitable for the expression of other small peptides.     

On-off regulation of gene expression from tryptophan promoter with cross-flow filtration

Summary Recombinant plasmid containing β-galactosidase gene fused to trp promoter (pMCT98) and that containing cloned trp repressor gene (pRLK13) were introduced into Escherichia coli C600. The bacterium was cultivated in a jar-fermetor equipped with a cross-flow filtration apparatus to attain the on-off regulation of the gene expression by controlling tryptophan concentration in the medium. In logarithmic growth phase, the cross-flow filtration was started. Tryptophan concentration dropped to a low level within 1 h and an efficient expression of β-galactosidase gene was started. By this twostage cultivation, very high biomass was achieved (final OD₅₇₀: 150) and the amount of produced β-galactosidase was about 10% of total cellular proteins.     

Factors affecting the expression of foreign proteins inEscherichia coli

Summary A variety of factors affect the expression of foreign proteins inEscherichia coli. These include: promoter strength, efficiency of ribosome binding, stability of the foreign protein inE. coli, location of the foreign protein inE. coli, the codons used to encode the foreign protein, the metabolic state of the cell, and the location, stability and copy number of the foreign gene. This paper contains a critical review of these factors with the idea that a detailed understanding of them is the key to the development of strategies for the efficient large-scale production of foreign proteins inE. coli.     

Formation of recombinants inEscherichia coli and a gene controlling the expression of a donor marker

In our experiments we tried to explain some anomalies in the formation of recombinants in anEscherichia coli mutant strain which, compared with the control, yields a lower number of recombinants. Following the transfer of C¹⁴-thymidine-labelled donor chromosome it was found that the low recombinant frequency is not due to a lower effectiveness of DNA transfer into the recipient cell; similarly, using the technique of interrupted mating between the cells we were able to detect that the rate of chromosome transfer is the same as in the control. The low frequency of recombinants may be explained by some restriction processes which take place in the recipient cell following the chromosome transfer. In addition, a relation between the localization of a marker and its subsequent expression in the recombinants was observed. Analyzing this phenomenon we were able to find a gene located near thethr marker which might modify the expression of integrated donor marker in the zygote. The probable mode of action of this gene is discussed.