Maximizing the expression of a recombinant gene in Escherichia coli by manipulation of induction time using lactose as inducer.

The use of isopropyl-beta-D-thiogalactoside (IPTG) for induction of the lac-promoter in small-scale cultivations is well established. However, for large-scale microbiological processes the cost of this inducer is a severe limitation. Here is described a method by which lactose is used as inducer of the lac promoter with the same efficiency as that of IPTG. It was found that after growth on glucose the time of the addition of lactose is important for the quality of induction. The resulting yield of the recombinant protein increased when lactose was added to the culture if the glucose concentration was rather low. By careful monitoring of the glucose level in the fermentation, using a biosensor, it was possible to add the inducer when the carbon source was nearly depleted. Using Escherichia coli BL21 (pET3), in which was cloned the main antigen coat protein of the foot and mouth disease virus, induction of the gene led to expression of the target protein at a level exceeding 20% of the total cell protein.

cDNA-cloning and expression of VP1-specific sequences of foot-and-mouth disease virus types A5 and O1.

The RNA genome of foot- and mouth disease virus strains A5 Westerwald and O1 Lausanne has been reverse-transcribed and cloned in lambdaphages or plasmids. Identification of cDNA-clones containing VP1-specific sequences was achieved by hybridization, restriction mapping, and sequence analysis. VP1-coding cDNA-fragments were subcloned into the expression vector pEX which led to synthesis of fusion proteins with beta-galactosidase. These fusion proteins reacted with anti-VP1 antibodies on a Western blot, but were not capable of inducing neutralizing antibodies to mice. This seemed to suggest a tertiary structure of the VP1-epitopes unlike those of native VP1. Other attempts are discussed to construct VP1-fusion proteins folding similarly to the native viral protein structure.

[Structure of the enzootic cattle leukosis virus. 1. Biophysicochemical characterization of several virus-specific components]. / Struktur des Virus der enzootischen Rinderleukose. 1. Mitteilung: Biophysikochemische Charkaterisierung mehrerer virusspezifischer Komponenten.

Concentrated virus material partially purified, after having been obtained from permanent cell cultures, was allowed to undergo repeated gradient centrifugation for an analysis of density and sedimentation. Serological tests were conducted, in that context, together with electron microscopy and gel electrophoresis. Isodensities of the virion were 1.17 g/ml in CsCl solution or 1.16 g/ml in saccharose solution. The average densities of the internal bodies, between 45 nm and 80 nm in diameter, were measured by electron microscopy and accounted for something between 1.32 g/ml and 1.34 g/ml in CsCl solution or between 1.22 g/ml and 1.24 g/ml up to 1.28 g/ml in saccharose solution. Four revertase-positive components, 915 S, 704 S, 469 S and 264 S +/- 40 S, were found in flattened cane-sugar gradients. conclusions for non-invasive ultracentrifugation in the virus were drawn from those high S-values. High-titre mixed antigens, virus-specific bands from the density gradients, and washed antigen-antibody precipitates were investigated by the authors by means of slab-gel electrophoresis. Molecular weights of 53 kilodalton (kD) and 24 kD were recorded from the major components. There also were polypeptides weighing 10 kD, 12 kD, 15.6 kD, 42.5 kD, 63 kD, and 70 kD.