Mapping of an epitope of human leukocyte alpha interferon A which is recognized by the murine monoclonal antibody NK2.

An epitope of human leukocyte alpha interferon A (IFN-A), which is recognized by the murine monoclonal antibody NK2, has been mapped by using four successive approaches. Limited proteolysis of the IFN-A chain, followed by electrophoresis, Western blotting, and probing of the proteolytic fragments with NK2 showed that an epitope was located within the sequence residues 110-140. A panel of human IFN subtypes bearing substitutions within the sequence 110-140 was tested for reactivity with NK2 in enzyme-linked immunosorbent assays.

Temperature-dependent activation of Bacillus brevis metalloprotease expressed in mesophilic Bacillus subtilis.

When the Bacillus brevis secretory metalloprotease is expressed from the npr gene on a plasmid vector in the mesophile B. subtilis, grown at 37 degrees C, the enzyme was found to be properly processed, but secreted into the culture medium in a low-active conformation. Secreted metalloprotease can by heat-treatment (70 degrees C for 30 min) be converted into fully active enzyme.

Cloning and expression of Clostridium thermocellum genes coding for thermostable exoglucanases (cellobiohydrolases) in Escherichia coli cells.

By special screening approach two independent Cl. thermocellum genes directing the synthesis of thermostable glucanases with an exo-mode of action have been isolated from pUC19-based gene bank in E. coli TG1.

Reconstruction of an epitope capable of binding murine monoclonal antibodies NK2 within the sequence of human leukocyte interferon alpha F by site-directed mutagenesis.

Using site-directed mutagenesis, an epitope of human leukocyte IFN-A capable of binding murine monoclonal antibodies NK2 has been reconstructed within the sequence of IFN-F, which lacked ability to bind these particular type of antibodies. It was found by the analysis of the recombinant IFN-A1-92/F93-100 encoding by a hybrid gene that N-terminal part of IFN-A played no significant role in NK2 binding. Then by site-directed mutagenesis of IFN-F gene three residues of IFN-F polypeptide chain (Asn113, Val114 and Lys121) were substituted for the residues of IFN-A sequence (Lys113, Glu114 and Arg121), and it was shown that the mutated IFN-F(A 113, 114) and IFN-F(A 113, 114, 121) restored the partial and full ability, respectively, to bind NK2 as compared with unmodified IFN-A, while IFN-F(A 121) lacked NK2 binding completely, as well as parental IFN-F.

The intrinsic fluorescence of the recombinant human leukocyte interferon-alpha A and fibroblast interferon-beta 1.

The environment of Trp residues of the recombinant human interferons has been studied by the analysis of the emission spectra of native and denatured proteins at pH 1.5-8.5 and temperature 10-65 degrees C in the presence and absence of the anionic, cationic and neutral to charge contact quenchers--KI, CsCl and acrylamide, respectively.

Bacillus amyloliquefaciens alpha-amylase signal sequence fused in frame with human proinsulin is properly processed by Bacillus subtilis cells.

The plasmid pBINS1, containing the promoter, SD and leader peptide sequences of Bacillus amyloliquefaciens alpha-amylase gene and 267 bp long sequence coding for human proinsulin directs the efficient synthesis of hybrid preproinsulin, as well as quantitative secretion of proinsulin outside of protease-deficient Bacillus subtilis AJ73 cells. The recombinant proinsulin has been isolated from the culture medium and its N-terminal sequence shown to be identical with that of natural human prohormone.

Cloning of Clostridium thermocellum endoglucanase genes in Escherichia coli.

Six independent and distinct cel genes coding endoglucanases have been selected from C. thermocellum pUC19-based gene bank in E. coli TG1.

Chemical modification of the recombinant human alpha A- and beta-interferons.

Chemical modification has been used to map the residues essential for the antiviral activity of the recombinant human alpha A- and beta-interferons. Modification of His residues with diethylpyrocarbonate and N alpha-tosyl-L-lysyl chloromethylketone does not inhibit both interferons, whereas N alpha-tosyl-L-phenylalanyl chloromethylketone significantly suppressing the activity of beta-interferon does not affect the activity of alpha A-interferon. After the modification of 1, 2 and 3 Lys residues from 11 ones with 3-(2-pyridyldithio)propionic acid N-hydroxy-succinimide ester alpha A-interferon reveals 100%, 50% and 10% of the initial activity, respectively.

The isolation and properties of collagenolytic proteases from crab hepatopancreas.

A mixture of collagenolytic proteases has been isolated from the Kamchatka crab hepatopancreas. The four individual enzymes were further separated with FPLC and partially characterized. Crab collagenolytic proteases possess a high activity against different types of collagen, especially against calf skin collagen Type III and bovine lens capsule collagen Type IV, which is resistant to the microbial Clostridium sp.