Expression of repetitive human calcitonin genes in Escherichia coli.

In order to stabilize recombinant human calcitonin (rhCT) against Escherichia coli proteases a series of concatemeric hCT genes with varying degrees of repetition were synthesized and expressed in E. coli under the control of a constitutive synthetic phage promoter. The series of expression vectors thus constructed was used as a model to study the effect of gene repetition on the efficiency of expression (both transcription and translation), stability of mRNA, proteolytic stability of recombinant protein, genetic stability of expression plasmids, etc. The oligomerization of the hCT gene resulted in stabilization of the mRNA increasing its half-life from 60-70 s (as in the hCT monomer, dimer, and trimer genes) to 100-120 s (for the hCT tetramer gene). This effect held true as well for the proteins coded by the corresponding repetitive hCT genes. The genetic stability (segregation and recombination) of the expression plasmids containing hCT oligomeric genes also depended on the number of hCT gene repeats. The expression plasmid containing the hCT tetramer gene segregated from one of the best producers of rhCT (E. coli LE392) up to 100% after 100 cell generations in nonselective media (free of antibiotics). One of the plasmids most sensitive to recombination events was that containing the hCT pentamer gene. The series of expression plasmids bearing hCT oligomeric genes was used for transformation of various E. coli strains in order to find the optimal host for production of rhCT. The highest yield (44-100 mg rhCT per 1 liter of bacterial culture) was obtained with the strains LE392, JM107, and DH1.

Association of actin with DNA and nuclear matrix from Guerin ascites tumour cells.

The protein composition of nuclear matrices containing different amount of DNA was examined. It was found that, in matrices containing 2% to 80% of total DNA, the quantity of DNA-bound proteins remains relatively constant varying from 10% to 15% of total nuclear proteins. Electrophoretic patterns do not differ substantially, but autoradiograms with in vitro 125I labelled proteins show quantitative variations in the actin content. Application of radioimmunoassay (RIA) enabled to determine the exact content of actin in GAT nuclei and nuclear matrices - 5 micrograms/ml in nuclei, of which 50% are bound to DNA and 30% being a component of the protein part of the nuclear matrix. These results are supported by electron microscopic data, where immunogold technique was performed on thin sections and spread material. The applied methods suggest that part of the nuclear actin is tightly bound (resistant to 2 M NaCl) to DNA and represents a component of the internal nuclear matrix.

Expression of synthetic calcitonin genes in plasmid vectors containing tandemly repeated non-overlapping ribosome binding sites.

1. Expression plasmids containing non-overlapping tandemly repeated ribosome binding sites (RBS) were constructed in order to stabilize mRNA and enhance translation. 2. Two synthetic genes (human calcitonin tetramer gene and a fusion gene human gamma-interferon-human calcitonin) were cloned in these vectors and the effect of multiplicity of Shine-Dalgarno (S/D) sequence on heterologous gene expression was studied. 3. It was found that duplication and triplication of RBS had no effect on the stability of mRNA but led to a strong decrease in the level of recombinant protein and mRNA in the cell. 4. Plasmids bearing four times repeated S/D sequences gave longer-lived mRNAs and maintained a level of protein and mRNA very close to the values obtained with a single S/D containing plasmids.

Possible synthesis of steroid-protein for immunization with a fixed narrow range of the hapten-protein ratio.

A comparison of two methods for synthesis of a steroid-protein conjugate (progesterone-11 alpha-hemisuccinate-BSA) has been made. Under the conditions of the method described, using tetrahydrofurane as a medium for the coupling reaction, stable intermediate products were obtained. The resulting conjugate had a narrow range of the hapten-protein ratio (18-20 steroid molecules per molecule of BSA), very good solubility in water and almost no unreacted BSA in the sample. Antisera raised against this conjugate, applied in low dose (50 micrograms), had quite satisfactory characteristics concerning their titre and specificity. The tests for recovery, sensitivity and the range of measurement established the possibility of using such antisera for the radioimmunoassay of progesterone.

The effect of the purity of the iodinated tracer on the specificity of a homologous assay of ovine follicle stimulating hormone.

Six different chromatographic procedures were employed to separate the intact labelled ovine follicle stimulating hormone after iodination of a highly purified preparation. The immunoreactivity of the fractions was tested in the conditions of a double-antibody radioimmunoassay. A single point crossreaction was introduced to calculate the interference of luteinizing hormone in the assay. The results obtained after SDS electrophoresis of the hormone were compared with the results of the autoradiography of the fractions subjected to electrophoresis after labelling and purification. Intact hormone with both subunits present was recovered following chromatography on Blue Sepharose C1-6B or high resolution gel filtration on Ultrogel AcA-54.