Use of proZZ-obelin fusion protein in bioluminescent immunoassay.

Obelin is a photoprotein that emits light by Ca2+-binding. To develop a bioluminescent immunoassay based on the light emission property of obelin, we have expressed the apoobelin fusion protein with ZZ-domain of S. aureus protein A in E. coli by recombinant DNA techniques. The proZZ-obelin expressed was purified by one-step affinity chromatography on IgG-Agarose. The purified proZZ-obelin has both the luminescent activity of obelin and the IgG-binding ability of ZZ-domain. The specific activity of fusion protein was 8.5 X 10(15) photons per mg of protein.

Sequence of the cDNA encoding the Ca(2+)-activated photoprotein obelin from the hydroid polyp Obelia longissima.

A cDNA clone encoding the Ca(2+)-activated photoprotein, obelin (Obl), from Obelia longissima was sequenced. The nucleotide (nt) sequence contained two long overlapping open reading frames (ORFs), one of which encoded apoobelin (apoObl). The deduced amino acid (aa) sequence of apoObl revealed that this 195-aa protein has three EF-hand structures that are characteristic for Ca(2+)-binding domains. Strong aa homology was shown among apoObl, apoaequorin and apoclytin. The second ORF present in the obl cDNA consists of 139 codons and encodes a very basic protein with a calculated pI of 10.56 and a molecular mass of 16,153 Da.

Expression and properties of the recombinant lumazine (riboflavin) protein from Photobacterium leiognathi.

Photobacterium leiognathi lumazine protein has been expressed in Escherichia coli in high yield, 30 mg/l. The cloned gene was one previously reported by Illarionov (EMBL X56534), that had a similar sequence and was located in the same position as the lumazine protein gene in P. phosphoreum. This gene was placed downstream of the T7 gene 10 promoter of the plasmid pT7-7. When the E. coli are grown at 37 degrees C the protein accumulates in inclusion bodies but solubilization can be achieved in 6 M urea. By a simple procedure of dialysis in the presence of riboflavin and centrifugation, without any chromatography, the recombinant holoprotein is purified to 95% homogeneity. The spectral properties of this recombinant riboflavin protein are the same as those of a fluorescent riboflavin-bound protein produced by many strains of P. leiognathi. The absorption spectrum has the same maxima, 276, 386, 464 nm, the circular dichroism is also the same, and both absorption spectrum and CD are the same as that of apo-lumazine protein having riboflavin bound. The riboflavin on the recombinant can be easily replaced by 6,7-dimethyl-8-ribityllumazine. The absorption and fluorescence spectra, fluorescence yield, and bioluminescence properties of this rebound protein identify it as authentic lumazine protein.

[A study of native streptokinase and its polymeric derivative by means of small angle x-ray scattering]. / Issledovanie nativnoi streptokinazy i ee polimernogo proizvodnogo metodom rentgenovskogo rasseianiia pod malymi uglami.

The gyration radius (R0) of native streptokinase (SK) was found to be R0 = (40 +/- ) A by small-angle X-ray scattering. Experimental hydrodynamic characteristics of SK were S0(20),W = (2.8 +/- 0.1)S; D0(20),W = (6.0 +/- 0.5) x 10(-7) cm2/s; [n] = 0.12 dl/g. The molecular weight of the enzyme was found to be 44,000. The values of the form factor R0/Rsphere = 2.1 and the frictional ratio f/f0 = 1.5 indicate considerable anisometry of the SK molecule. Basing on the curves of small-angle X-ray scattering of SK modified with a synthetic linear copolymer of N-vinylpyrrolidone (P) at a molar ratio SK less than P, a structural model of the conjugate was proposed. The modified form consisted of a dense nucleus covered with a diffuse polymeric membrane. In accordance with the model, R0 of modified SK and of the whole conjugate were found to be R0nucleus = (34 +/- 2) A and R0conjugate = (114 +/- 5)A.

[Effect of emulsifiers released by yeasts multiplying on n-alkane media on their growth and microscopic organization]. / Vliianie émul'gatorov, vydeliaemykh drozhzhami, razmnozhaiushchimisia na sredakh s n-alkanami, na ikh rost i mikroskopicheskuiu organizatsiiu.

Bioemulsifiers and ether extracts from the cultural broth of yeast cells were used to study their effect of Candida lipolytica growth and ultrastructural organisation. When the emulsifiers and ether extracts were added to the growth medium, the lag phase was reduced but the growth rate did not change. The ether extracts increased the growth rate of C. lipolytica 374/2 and the final biomass yield of C. lipolytica 704. The ultrastructural organisation of C. lipolytica 374/2 cells changed under the action of the bioemulsifier added to the growth medium.

[Emulsifying activity of yeasts growing on normal alkanes]. / Emul'giruiushchaia aktivnost' drozhzhei pri roste na normal'nykh alkanakh.

The ability to emulsify n-hexadecane was compared among ten strains of Candida lipolytica. The cultures were shown to differ in the activity of emulsification. The highest activity was recorded in the phase of growth deceleration. Substances involved in n-alkane emulsification were isolated.

[Biosynthesis of citric and isocitric acids by the wild-type and mutant strains of Candida lipolytica in media containing different carbon sources]. / Biosintez limonnoi i izolimonnoi kislot prirodnym i mutantnym shtammami Candida lipolytica na sredakh s razlichnymi istochnikami ugleroda.

Experiments were carried out to examine the capacity of two strains of Candida lipolytica, producing citric and isocitric acids in the alkane and glucose containing media, to grow on different two- and three-carbon compounds. The strains did not grow on oxalate, glyoxalate, glycolate, malonate or propionate. When cultivated in the media containing acetate, ethanol, glycerol, glucose or hexadecane, supersynthesis of the acids started after complete consumption of the nitrogen source and resultant delay of the culture growth. Either strain discharged the two acids in a proportion that depended on the strain nature and the type of the carbon source. The mutant strain produced only citrate while the wild-type synthesized both citrate and isocitrate, the ratio of which was related to the nature of the carbon source utilized.

[Effect of cultivation conditions on the synthesis of citric and isocitric acids in Candida lipolytica on hexadecane medium]. / Izuchenie vliianiia uslovii kul'tivirovaniia na obrazovanie limonnoi i izolimonnoi kislot Candida lipolytica na srede s geksadekanom

The influence of aeration, pH and iron concentration on the growth of yeast C. lipolytica 704 on the hexadecane medium and on the synthesis of citric and isocitric acids was investigated. The yeast synthesized citric acids actively during intensive aeration. The acid formation was strongly dependent on the medium acidity: pH 6.0 was most favourable for the synthesis of citric acids. The Fe concentration influenced significantly the ratio of the acids synthesized. At a low concentration of iron (0.005 mg Fe/l) equal amounts of citrate and isocitrate were formed; at an increased concentration isocitrate was in predominant formation.