Expression of recombinant DNA functional products in Escherichia coli anucleate minicells

This review covers the use of anucleate minicells of Escherichia coli for expressing the recombinant DNA encoded proteins. We briefly discuss the methods being used for preparation of anucleate minicells, incorporation of cloned DNA and assessment of gene expression. While the largest use has been that of microbially derived cloned functional DNA, examples of eukaryotic gene product synthesis have also been reviewed. This technology may represent some interesting commercial opportunities.     

Minicell-forming mutants of Escherichia coli: production of minicells and anucleate rods.

The Escherichia coli minB mutant originally isolated is known to septate at cell poles to form spherical anucleate minicells. Three new minicell-producing mutants were isolated during a screening by autoradiography for chromosome partition mutants giving rise spontaneously to normal-sized anucleate cells. These min mutants were affected close to or in the minB locus. Autoradiography analysis as well as fluorescent staining of DNA showed that in addition to minicells, these strains and the original minB mutant also spontaneously produced anucleate rods of normal size and had an abnormal DNA distribution in filaments. These aberrations were not associated with spontaneous induction of the SOS response. Inhibition of DNA synthesis in these mutants gave rise to anucleate cells whose size was longer than unit cell length, suggesting that the min defect allows septation to take place at normally forbidden sites not only at cell poles but also far from poles. Abnormal DNA distribution and production of anucleate rods suggest that the Min product(s) could be involved in DNA distribution.     

Thymidine phosphorylase from Escherichia coli: tight-binding inhibitors as enzyme active-site titrants

Thymidine phosphorylase (EC 2.4.2.4) catalyses the reversible phosphorolysis of pyrimidine 2'-deoxynucleosides, forming 2-deoxyribose-1-phosphate and pyrimidine. 5-Chloro-6-(2-imino-pyrrolidin-1-yl)methyl-uracil hydrochloride (TPI, 1) and its 5-bromo analogue (2), 6-(2-amino-imidazol-1-yl)methyl-5-bromo-uracil (3) and its 5-chloro analogue (4) act as tight-binding stoichiometric inhibitors of recombinant E. coli thymidine phosphorylase, and thus can be used as the first active-site titrants for it using either thymidine or 5-nitro-2'-deoxyuridine as substrate.     

Change in the viscoelastic properties of agarose gel by HAp precipitation by osteoblasts cultured in an agarose gel matrix

The viscoelastic properties of cell-seeded agarose gel were measured as a function of culture time. Because the seeded cells, MC3T3-E1, were osteoblast-like cells, the system can be regarded as a model osteogenesis system. For all specimens the characteristic stress relaxation curve of agarose gel was observed—a large relaxation up to 10⁴ s followed by a gel plateau, where the former was attributed to molecular motion of polymer chains between two adjacent cross-links of the gel and the latter to the elasticity of the gel network. The viscoelasticity was quantified by fitting stress relaxation data to an empirical equation. The relaxation time and its distribution did not change with culture time. The initial and equilibrium moduli, E ₀ and E _e, respectively, and relaxation strength, ΔE = E ₀ ? E _e, did not change up to day 15 of culture but changed significantly at day 18 of culture. The change in ΔE with culture period correlated well with that in E ₀. The changes in the mechanical properties of the cell-seeded agarose gel system were explained in terms of the function of MC3T3-E1 in precipitating calcium phosphate mineral particles. The precipitation was detected by alizarin red S staining of the system at day 9 of culture. The precipitated calcium phosphate was confirmed to be hydroxyapatite (HAp) and the amount of HAp increased monotonically with culturing time, both of which were observed by X-ray diffraction studies. The dependence of the modulus of the composite on mineral fraction is discussed. A simple model of mixing of the components based on the continuum material concept was not applicable, but a model considering percolation of mineral particles in a network chain with culture time was suitable to explain the observed results. The results may be particularly important for predicting the stiffness of functionally engineered bony tissue implanted in a fractured bone.     

Nucleoside triphosphate pools in minicells of Escherichia coli.

The nucleoside triphosphate pools of Escherichia coli minicells are different from those in parental cells. The growth phase in which minicells accumulate significantly affects the pool sizes.     

Effect of pericellular matrix formation by chondrocytes cultured in agarose gel on the viscoelastic properties of agarose gel matrix

The viscoelasticity of chondrocyte-seeded agarose gel (AGC0) and that of chondrocyte-seeded agarose gel after 21 days of cultivation (AGC3) were investigated. In AGC3, pericellular matrix (PCM)-like material around each chondrocyte was found to be constructed, which was confirmed by an optical micrograph in conjunction with toluidine blue staining. The relaxation modulus of each of the chondrocyte-agarose gel composite systems was measured by a non-constrained indentation method. Stress-strain curves for all of the specimens examined had a toe region followed by a linear region terminated by specimen fracture. The slope of the linear region of AGC0 was smaller than that of AG, while the SS curve of AGC0 was indistinguishable from that of AGC3. All of the relaxation curves studied were typical of gels, having a fast relaxation process up to 10³ s followed by a plateau. The relaxation modulus of AGC0 was smaller than that of agarose gel (AG), the decrement in relaxation modulus from AG to AGC0 being attributed to the seeding of chondrocytes that have a smaller modulus than that of agarose gels. However, the relaxation modulus of AGC3 was increased at the early viscoelastic region in particular, as compared with that of AGC0. The increments in the relaxation modulus in AGC3 were attributed to the PCM-like material produced by chondrocytes, where the produced material may provide crosslink points and reinforce the agarose gel.     

Protective action of antioxidants on Escherichia coli cells immobilized in polyacrylamide gel

The object of this work was to find out whether antioxidants could be used for weakening the effect of free radicals on Escherichia coli cells immobilized in polyacrylamide gel. Some of the antioxidants soluble in lipids and water (ionol, Epigid, glutathione) protected the cells against the action of free radicals produced in the process of acrylamide polymerization, and increased the viability of the immobilized bacteria.     

Mucopeptide biosynthesis by minicells of Escherichia coli.

Minicells produced by Escherichia coli chi925 incorporated amino acids and N-acetyl-d-glucosamine into mucopeptide.     

Properties of cellulase immobilized on agarose gel with spacer

Cellulase produced by fungus Trichoderma viride was immobilized on agarose beads (Sepharose 4B) activated by cyanogen bromide and also on activated agarose beads that contained spacer arm (activated CH-Sepharose 4B and Affi-Gel 15). The CMCase activity retained by immobilized cellulase on activated Sepharose containing the spacer tended to be higher than that immobilized without spacer, although the extent of protein immobilization was lower. Also, the higher substrate specificity for cellulase immobilized on beads with spacer was obtained for cellobiose, acid-swollen cellulose, or cellulose powder. The hydrolysis product from their substrates was mainly glucose.     

Escherichia coli Hfr-DNA degradation in endonuclease I-deficient minicells.

[3H]Thymidine (dThd)-labelled Hfr DNA was transferred by conjugation into Escherichia coli F- minicells harvested from an endonuclease I-deficient (endI-) strain and its iosgenic wild type (endI+) parent. The susceptibility of this DNA to attack by DNAase was examined. The kinetics of in vivo conversion of [3H]dThd-labelled DNA into acid soluble radioactivity was examined. This activity, attributed to exonuclease action was the same for both strains. Contribution of endonuclease I was measured by an analysis of changes in weight-average (Mw) and number-average (Mn) molecular weight distribution of DNA molecules recovered from minicells. Reduction in Mw was greater in the endI-strain. The ratio Mn/Mw changed drastically during the incubation period of endI- minicells, but remained unchanged in the endI+ strain. These experiments suggest that the presence of the endI- mutation in minicell-producing strain chi1268 leads to a greater loss in M2 of Hfr DNA conjugally transferred into the minicells.     

Comparison of the aspartase activity and its stability in Escherichia coli cells immobilized on polyacrylamide gel and carrageenan

The conditions for immobilization of Escherichia coli cells (Soviet strain 85) on the natural polysaccharide carrier carrageenan (Soviet-made) were investigated and kinetic regularities of the aspartase reaction catalysed by immobilized in carrageenan cells of E. coli 85 were established. The conditions for retaining a high aspartase activity and stability of biocatalysts based on the E. coli 85 cells immobilized in PAAG and carrageenan were determined using full-loaded tanks for continuous synthesis of L-aspartic acid. The time-stable aspartase activity of the biocatalyst can be increased by treating the beads of the catalyst with bifunctional reagents (hexamethylenediamine, glutaraldehyde), the most active catalyst for the biotechnological synthesis of L-aspartic acid being obtained when carrageenan is used.     

Specificity and Efficiency of Thymidine Incorporation in Escherichia coli Lacking Thymidine Phosphorylase

A mutant of Escherichia coli lacking the catabolic enzyme thymidine phosphorylase readily incorporates exogenous thymidine into deoxyribonucleic acid (DNA) even when provided at concentrations as low as 0.2 mug/ml. Incorporation by this prototrophic strain occurs specifically into DNA, since, with radioactively labeled thymidine, (i) more than 98% is incorporated into alkali-stable material, (ii) at least 90% is recovered as thymine after brief formic acid hydrolysis, and (iii) at least 90% is incorporated into material with the buoyant density of DNA. During growth in medium containing thymidine, the bacteria obtain approximately half of their DNA thymines from the exogenous thymidine and half from endogenous synthesis. The thymines and cytosines of DNA can be simultaneously and specifically labeled by thymidine-2-(14)C and uridine-5-(3)H, respectively. The mutant, which does not degrade thymidine, retains the ability to degrade the thymidine analogue 5-bromodeoxyuridine.     

Evaluation of the activity of hydrolases and their inhibitors using substrates immobilized on agarose gel

A simple and convenient method for the quantitative evaluation of the activity of hydrolytic enzymes and their inhibitors has been proposed. This method is based on the immobilization of a substrate on agarose gel and the following evaluation of the enzyme activity by measuring the hydrolysis area around the well containing the enzyme solution. The method was shown to be applied to the evaluation of the activity of proteinases and amylases and the inhibitors of these enzymes in different biological objects.     

Failure to trigger the autolytic enzymes in minicells of Escherichia coli

Minicells from Escherichia coli P678-54 are refractory towards procedures known to induce bacteriolysis of DNA-containing E. coli cells. Although still engaged in murein synthesis, minicells could not be lysed by penicillin G. Likewise, endogenous overproduction of the cloned soluble lytic transglycosylase, the predominant murein hydrolytic activity in E. coli, failed to lyse minicells. Furthermore, induction of the phage MS2 lysis protein, a hydrophobic protein assumed to trigger the autolytic system of the host, did not result in bacteriolysis. It is concluded that the murein hydrolases present in minicells are under a tight cellular control.