Mucoadhesive polyelectrolyte microparticles containing recombinant human insulin and its analogs aspart and lispro.

Microparticles containing recombinant human insulin and its analogs aspart and lispro were prepared using an alternate adsorption of chitosan and dextran sulfate from solutions onto microaggregates of protein-dextran sulfate insoluble complex. The following properties of polyelectrolyte hormone-containing microparticles were studied: pH stability, surface charge, mucoadhesive properties, Ca(2+) binding, degradation under the influence of proteases (trypsin, chymotrypsin). The influence of the self-association ability of encapsulated insulins on the form of protein releasing from microparticles was studied. Insulins aspart and lispro released from the microparticles as monomers were more liable to proteolysis than human insulin released as a hexamer. The combined effect of properties of polyelectrolyte microparticles and of encapsulated recombinant proteins on the bioavailability of insulin under peroral administration is discussed.

[DNA assay for genetically engineered pharmaceutical substances using the real-time PCR technique].

A real-time PCR procedure is proposed for assaying E. coli residual DNA in the pharmaceutical substance of human recombinant insulin. For the quantitative analysis of the DNA content, an amplification of fragments of the bla gene plasmid DNA and E. coli genomic DNA of the 16S RNA gene were used. The contents of plasmid and genomic DNA were detected both in intermediates at various stages of the insulin purification process and in the finished product.

[Nanocomplexes of recombinant proteins and polysialic acid: preparation, characteristics, and biological activity].

A preparation of nanocomplexes containing recombinant proteins (interferons alpha2b and beta1b, insulin, and human granulocyte colony stimulating factor) and natural polysialic acid (PSA) has been described. The incorporation of protein into the complex changes its electrophoretic mobility. Atomic force microscopy reveals the average size of 23-kD insulin complexes with PSA of 10-20 nm and demonstrates that more than 60% of glycopolymer molecules carry a single protein molecule. Experiments with cultured cells show that cytokines bound to polysialic acid retain their ability to regulate cell proliferation. Insulin bound to PSA has a prolonged hypoglycemic effect in vivo.

[A PCR-based semiquantitative assay of DNA impurities in recombinant protein preparations].

A semiquantitative assay of DNA impurities in preparations of human recombinant insulin is described. The assay is based on the detection of a fragment of the ampicillin-resistant gene within the producer strain DNA by PCR. The analysis of PCR products of the studied preparations and PCR products containing known amounts of E. coli total DNA enabled a quantitative determination of the producer strain DNA content in the preparations under study. The sensitivity of the method is 7 pg of E. coli DNA per 10 microg of human recombinant insulin. The high sensitivity of the method allows us to recommend it for the quantitative determination of DNA content in recombinant preparations that do not inhibit PCR.

[Enteropeptidase and its use for cleavage of chimeric proteins]. / Enteropeptidaza i ee primenenie dlia rasshepleniia khimernykh belkov.

Cleavage of different chimeric proteins after specific linker (Asp)4Lys by the highly purified enteropeptidase was investigated, proteins being were accumulated in inclusion bodies or secreted from the cell. Kinetic constants for enzymatic hydrolysis were obtained, indicating that the substrate binding depended mainly on the affinity to the linker peptide (Asp)4Lys. Conditions for the efficient cleavage of recombinant proteins with enteropeptidase are formulated.

[Hydrolysis of chimeric proteins by enteropeptidase at the specific linker (Asp)4Lys depending on refolding conditions]. / Osobennosti gidroliza khimernykh belkov énteropeptidazoi po spetsificheskomu linkeru (Asp)4Lys v zavisimosti ot uslovii refoldinga.

Refolding from inclusion bodies of chimeric proteins containing the enteropeptidase-specific linker (Asp)4Lys was carried out. It was shown that, depending on the refolding conditions, chimeric proteins function as substrates or inhibitors of the enteropeptidase. The efficiency of the enteropeptidase hydrolysis of chimeric proteins containing the (Asp)4Lys linker may depend not only on the amino acid sequence of the protein binding site for the enzyme but also on the site conformation.

[5-fluoro-tryptophan-containing N-terminal domain of the alpha-subunit of the Torpedo californica acetylcholine receptor: preparation in E. coli and 19F NMR study]. / Poluchenie v E. coli i 19F-IaMR issledovanei 5-ftortriptofansoderzhashchego n-kontsevogo domena alpha-sub''edinitsy atsetilcholinovogo retseptora Torpedo californica.

A protein corresponding to the extracellular 1-209 domain of the alpha-subunit of the nicotine acetylcholine receptor from the electric organ of Torpedo californica was prepared using the corresponding cDNA domain by culturing Escherichia coli cells on a synthetic medium supplemented with 5-fluoro-L-tryptophan. The presence of a (His)6 fragment preceding the 1-209 sequence allowed purification of the protein isolated from inclusion bodies by affinity chromatography on Ni-NTA Agarose. The incorporation of 5-fluorotryptophan residues was found by 19F NMR to be approximately 50%. The spectrum of the protein reduced under denaturing conditions and subsequently reoxidized in a dilute solution under denaturing conditions in the presence of 0.05% SDS was sufficiently resolved, which allowed partial assignment of 19F resonances using the Trp60Phe mutant protein. The ability of the prepared domains to specifically bind snake alpha-neurotoxins was demonstrated with the use of radioiodinated alpha-bungarotoxin and trifluoroacetylated alpha-cobratoxin.

[Inhibition by IFN-(Asp)4-Lys-HIV chimeric protein of hydrolysis of the low molecular substrate by the enteropeptidase light chain]. / Ingibirovanie khimernym belkom IFN-(Asp)4-Lys-HIV gidroliza nizkomolekuliarnogo substrata legkoi tsep'iu énteropeptidazy.

The full length enteropeptidase or it's light chain have often used for the limited proteolysis of recombinant chimeric proteins incorporating the linker-(Asp)4Lys- to obtain the target protein. Any chimeric proteins were not cleaved by the full length enteropeptidase efficiently. The resistant to the hydrolysis chimeric protein IFN-(Asp)4Lys-HIV earlier was shown to be the competitive inhibitor (Ki = 3,4 x 10(-6) M) in relation to the low molecular substrate. In present study we were determined this chimeric protein competitive inhibited the same substrate hydrolysis by enteropeptidase light chain (Ki = 2,7 x 10(-5) M). Comparison the Ki values for the substrate hydrolysis by full length enzyme and its light chain suggests that the enteropeptidase heavy chain may participate in chimeric protein binding.

[Formation of classical C3 convertase during the alternative pathway of human complement activation]. / Obrazovanie klassicheskoi C3-konvertazy v khode aktivatsii al'ternativnogo puti komplementa cheloveka.

The fluid phase C3 convertase of the alternative pathway of human complement activation has been constructed from the isolated C3 component and from purified factors B and D. The enzyme was able to activate the isolated components C4 and C2 in the presence of C4 but had no effect on C2 in the absence of C4. The C4 and C2 activation was monitored by the loss of their hemolytic activity during the incubation with the alternative fluid phase C3 convertase. The activation of C4 and C2 components by the membrane-bound alternative C3 convertase formed on red cells (EC3bBb) was followed by the formation of C3 convertase of the classic pathway--EC4b2a. This resulted in the enhancement of hemolysis.