Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Active 3C protease of poliovirus 1(M) was obtained when cloning and expressing fragment HindII-HindIII (bases from 5240 to 6056) of cDNA in vector pTTQ8 in E.coli cells. As shown, fragment 3D of polymerase covalently bound to 3C does not deprive the enzyme of its specific proteolytic activity. The absence of 26 N-terminal amino acids in 3C entails its inactivation. The recombinant 3C protease cleaved peptide bond Gln-Gly not only in virus polyprotein, but also in molecules of beta-galactosidase and bovine catalase.
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PMID:Recombinant poliovirus 3C protease. The enzyme application to protein specific fragmentation. 164 25

Alpha complementation of beta-galactosidase (beta gal) is intracistronic and requires interaction between the alpha donor region (residues 3-41) and alpha acceptor fragment (produced by M15). We have constructed two plasmids which direct the synthesis of hybrid beta gal: coxsackievirus proteins in Escherichia coli. One plasmid, pBD1045, encodes an enzymatically active 3C protease of coxsackievirus B3 fused between the amino-terminal 79 amino acids of beta gal (containing the alpha donor region) and amino acids 80 to 1023 (alpha acceptor region). A second plasmid, pBD1043 encodes an inactive 3C protease and results in a fusion of 260 coxsackievirus amino acids between residues 79 and 80 of the beta gal monomer. Both hybrid proteins expressed by these constructs have beta-galactosidase activity regardless of whether the viral protease (183 amino acids) is autocatalytically cleaved out of the chimeric protein (pBD1045) or remains as part of a fusion protein (pBD1043). The implications of these results for structural flexibility of the complemented beta-galactosidase enzyme are discussed.
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PMID:Expression of functional beta-galactosidase containing the coxsackievirus 3C protease as an internal fusion. 190 18

The RNA polymerase gene of human coronavirus (HCV) 229E encodes a large polyprotein that contains domains with motifs characteristic of both papain-like cysteine proteinases and proteinases with homology to the 3C proteinase of picornaviruses. In this study, we have, first, expressed the putative HCV 229E 3C-like proteinase domain as part of a beta-galactosidase fusion protein in Escherichia coli and have shown that the expressed protein has proteolytic activity. The substitution of one amino acid within the predicted proteinase domain (His-3006-->Asp-3006) abolishes, or at least significantly reduces, this activity. Amino-terminal sequence analysis of a purified, 34-kDa cleavage product shows that the bacterial fusion protein is cleaved at the dipeptide Gln-2965-Ala-2966, which is the predicted amino-terminal end of the putative 3C-like proteinase domain. Second, we have confirmed the proteolytic activity of a bacterially expressed polypeptide with the amino acid sequence of the predicted HCV 229E 3C-like proteinase by trans cleavage of an in vitro translated polypeptide encoded within open reading frame 1b of the RNA polymerase gene. Finally, using fusion protein-specific antiserum, we have identified a 34-kDa, 3C-like proteinase polypeptide in HCV 229E-infected MRC-5 cells. This polypeptide can be detected as early as 3 to 5 h postinfection but is present in the infected cell in very low amounts. These data contribute to the characterization of the 3C-like proteinase activity of HCV 229E.
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PMID:Characterization of a human coronavirus (strain 229E) 3C-like proteinase activity. 776 94

Expression and secretion of recombinant proteins in the endotoxin-free bacterium, Bacillus subtilis, has been thoroughly studied, but overexpression in the cytoplasm has been limited to only a few proteins. Here, we used the robust IPTG-inducible promoter, Pgrac212, to overexpress human rhinovirus 3C protease (HRV3C) in the cytoplasm of B. subtilis cells. A novel solubility tag, the N-terminal domain of the lysS gene of B. subtilis coding for a lysyl-tRNA synthetase was placed at the N terminus with a cleavage site for the endoprotease HRV3C, followed by His-HRV3C or His-GST-HRV3C. The recombinant protease was purified by using a Ni-NTA column. In this study, the His-HRV3C and His-GST-HRV3C proteases were overexpressed in the cytoplasm of B. subtilis at 11% and 16% of the total cellular proteins, respectively. The specific protease activities were 8065 U/mg for His-HRV3C and 3623 U/mg for His-GST-HRV3C. The purified enzymes were used to cleave two different substrates followed by purification of the two different protein targets, the green fluorescent protein and the beta-galactosidase. In conclusion, the combination of an inducible promoter Pgrac212 and a solubility tag allowed the overexpression of the HRV3C protease in the cytoplasm of B. subtilis. The resulting fusion protein was purified using a nickel column and was active in cleaving target proteins to remove the fusion tags. This study offers an effective method for producing recombinant proteins in the cytoplasm of endotoxin-free bacteria.
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PMID:Using the IPTG-Inducible Pgrac212 Promoter for Overexpression of Human Rhinovirus 3C Protease Fusions in the Cytoplasm of Bacillus subtilis Cells. 3161 59