Effects of temperature and novobiocin on the expression of calf prochymosin gene and on plasmid copy number in recombinant Escherichia coli.

Escherichia coli strain HB101 harboring an expression plasmid bearing calf prochymosin gene under the control of the tac promoter was grown in the presence of IPTG with or without novobiocin at 28 and 40 degrees C, respectively. The differential rates of synthesis of prochymosin inclusions, and, for comparison, of beta-lactamase and beta-galactosidase, as well as plasmid copy number, were determined during the first hours of steady state growth. At 28 degrees C the induced expression of prochymosin gene was almost blocked. Addition of novobiocin did not alleviate this effect. In fact, it strengthened it, and we conclude that both these additive inhibitory effects are a consequence of the decrease in negative superhelical tension of plasmid DNA to an insufficient level. At 40 degrees C the differential rate of prochymosin synthesis was markedly enhanced. Since the copy number of the expression plasmid increased approximately to the same extent, we conclude that an increase in gene dose is the cause. The stimulation of cloned heterologous gene expression at 40 degrees C and inhibition at 28 degrees C may be conveniently used in biotechnological-scale cultivations of some recombinant bacteria.

Improved procedure for a high-yield recovery of enzymatically active recombinant calf chymosin from Escherichia coli inclusion bodies.

The high-yield recovery of enzymatically active recombinant calf chymosin from Escherichia coli inclusion bodies was achieved by optimization of solubilization and renaturation conditions. The solubilization was carried out in 8 M urea at various pHs, at various temperatures, and for various periods of time. The following values were found optimal: 1 h at 31 degrees C, pH 10.4. For successful correct refolding of solubilized prochymosin molecules it was found to be necessary to dilute the solution into an alkaline buffer (pH 10.7) in such a way that the final concentration of urea did not exceed 0.32 M and that of protein 0.275 mg/ml. Our optimized procedure gives about eight times higher yields of enzymatically active chymosin than the current published methods.

Isolation, biochemical characterization and crystallization of the p15gag proteinase of myeloblastosis associated virus expressed in E. coli.

1. The p15gag proteinase responsible for the processing of the polyprotein precursor of the myeloblastosis associated virus was obtained by a recombinant technique in an E. coli expression system. The massive expression of the intentionally truncated precursor (Pr25lac-delta gag) was accompanied by its structurally correct processing. 2. Three procedures for the purification of the recombinant proteinase from both the cytoplasmic fraction and the inclusion bodies were developed. 3. The purified proteinase was compared with the authentic proteinase isolated from MAV virions by N-terminal sequence analysis and amino acid analysis, molecular weight determination, reverse-phase HPLC and FPLC elution profiles, electrophoretic mobility and isoelectric point determination, and activity assays with proteins and synthetic substrates. The identity of both enzymes was shown. 3. Contrary to reported data, the amino acid sequence of the p15gag proteinase differs from the sequence of the homologous Rous sarcoma virus proteinase in one residue only, as follows from cDNA sequencing. 4. Crystallization of the proteinase from a citrate-phosphate buffer at pH 5.6 afforded hexagonal crystals which diffracted well as 2.3 A without deterioration.

Subsite specificity of the proteinase from myeloblastosis associated virus.

The subsite requirements of the aspartic proteinase from the myeloblastosis-associated virus (MAV) for the cleavage of peptide substrates were studied with a series of synthetic peptides of general structure Ala-Thr-P4-P3-P2-P1*Nph-Val-Arg-Lys-Ala. The residues in positions P4, P3, P2 and P1 were varied and the kinetic parameters for the cleavage of substrates in 2.0 M NaCl were spectrophotometrically determined at pH 6.0 and 37 degrees C. The acceptance of amino acid residues in particular subsites is similar to that observed with the human immunodeficiency virus type 1 (HIV-1) proteinase in our earlier studies on the same substrate series: hydrophobic or aromatic residues are preferable in P1 position, a broad variety of residues are acceptable in P3 whereas the residues occupying P2 plays the decisive role in the substrate cleavage as evidenced by its dramatic influence on both kcat and Km values. The most remarkable difference between the two enzymes was found in P3 and P4 subsites. In P3, the introduction of negatively charged glutamate increases the substrate binding by the MAV proteinase 12-fold and decreases binding by the HIV-1 proteinase. In P4, Pro in this series is a favourable residue for the MAV proteinase and is strongly inacceptable for HIV-1 the proteinase. The pH profile of the cleavage was studied with a chromogenic substrate and differences between HIV-1 and MAV proteinases are discussed.

Fermentation conditions for high-level expression of the tac-promoter-controlled calf prochymosin cDNA in Escherichia coli HB101.

Escherichia coli HB101 harboring an expression plasmid that bears the calf prochymosin gene controlled by the tac promoter was cultivated under different conditions in order to find an optimal fermentation arrangement that would lead to maximal prochymosin yield. Our results indicate that it is advantageous to use lactose in the double role of inducer and carbon/energy source when foreign gene expression is controlled by the tac promoter and the gene product is only moderately toxic owing to its accumulation in the form of an intracellular body. Glucose, on the other hand, may be used when expression should be repressed. Growth temperature substantially influenced the specific rate of prochymosin and beta-lactamase gene expression and the plasmid copy number. Three phases were distinguished in the time course of the fermentation on lactose: exponential growth practically without prochymosin synthesis, linear growth with prochymosin synthesis, and prochymosin synthesis without growth of biomass. The synthesis of prochymosin in the form of intracellular inclusion body was accompanied by the loss of respiratory activity of the cell and the loss of its ability to multiply. Sixteen hours cultivation at 37 degrees C in a complex medium with lactose as inducer and carbon/energy source resulted in up to 30% of the volume and 48% of the total protein of biomass being accumulated for as prochymosin inclusion bodies. The concentration of extractable enzymatically active chymosin in the culture reached 12 mg/L.

Processed enzymatically active protease (p15gag) of avian retrovirus obtained in an E. coli system expressing a recombinant precursor (Pr25lac-delta gag).

Processing proteases of avian and mammalian retroviruses cut the polyprotein precursors encoded by the retroviral genes into mature functional proteins. Retroviral processing proteases are still a rather poorly characterized group as to their relation to other proteases, specificity, and mechanism of enzymatic action. In avian retroviruses the generation of the processing protease itself comprises a processing cleavage event - the protease p15gag is cut off the carboxy-terminus of a gag polyprotein precursor, Pr76gag. We report here that direct and efficient production of the avian retrovirus processing protease p15gag (required for structure-function studies and rational design of inhibitors) was obtained in an E. coli system, where massive expression of a size-reduced, recombinant precursor (Pr25lac-delta gag) was accompanied by its structurally accurate processing.

Two sites of oxygen control in induced synthesis of respiratory nitrate reductase in Escherichia coli.

The time course of appearance of respiratory nitrate reductase in Escherichia coli after induction by nitrate was analyzed under different conditions, and the inhibitory effects of oxygen, chloramphenicol, and rifampin were compared. Oxygen appeared to inhibit the synthesis of nitrate reductase at the level of transcription. In addition, the translation or some later steps of enzyme formation were blocked.

Effect of glucose on the biochemical properties of the bacterial cytoplasmic membrane.

The cytoplasmic membrane isolated from cells of Citrobacter freundii growing in a cultivation medium with glucose was found to exhibit a decreased ability to oxidize formate and succinate and a decreased activity of formate, succinate and lactate dehydrogenase as compared with the cytoplasmic membrane of cells growing on galactose. The activation energy for dehydrogenation and oxidation of succinate simultaneously increases. The quantitative content of cytochromes and quinones in both types of membranes does not differ considerably. The two types of membranes also do not mutually differ in the qualitative and quantitative representation of fatty acids in lipids and in the sensitivity to the effect of low concentrations of detergents. The mass ratio of proteins and lipids is lower in the membranes of cells grown on glucose.

Participation of quinone and cytochrome b in tetrathionate reductase respiratory chain of Citrobacter freundii.

Formate dehydrogenase, NADH dehydrogenase, a quinone and a b-type cytochrome characterized by maxima at 429 and 560 nm are shown to participate in the tetrathionate redox chain of Citrobacter.

Physiological characteristics of chemostatically grown Citrobacter freundii as a function of the specific growth rate and type of nutrient limitation.

Citrobacter freundii was grown aerobically in a chemostat on a mineral medium with galactose or glucose as carbon and energy sources under limitation by carbon or nitrogen source respectively. At various specific growth rates ranging from 7 to 95% mumax the culture in steady state was analysed and growth yield, specific metabolic rate of substrate utilization, intracellular concentration of pyruvate, ATP, ADP, AMP and energy charge were determined and plotted as functions of dilution rate. In all four types of experiments the physiological state of cells remained practically independent of dilution rate up to D=0.6 mumax, and at a given specific growth rate nearly independent on mumax and type of limitation. At approximately D=0.6 mumax, which is close to the maximum output dilution rate Dm, the physiological state of the cells changed: growth yields decreased and intr cellular pyruvate and adenylates concentrations increased. Consequently, in a given medium two dilution rates exist at which growth rate dx/dt is the same but the physiology of the population is quite different.