Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The tolerance of bacteriophage lambda morphogenesis for C-terminal additions to the tail tube major protein subunit (the V gene product; gpV) has been investigated. A second modified copy of the lambda V gene, either within a novel phage vector itself or plasmid-borne, was expressed during phage growth. High-level substitution of wild-type gpV by modified gpV bearing a basic C-terminal peptide sequence (RRASV; a target site for cAMP-dependent protein kinase) was possible using multiple repeats of a serine-glycine (SGGG) linker sequence. Highly purified phage bearing copies of gpV-RRASV could be efficiently phosphorylated by the appropriate protein kinase, and the incorporated label was shown to migrate exclusively at the expected size in protein gels. A large tetrameric protein (beta-galactosidase) could be incorporated into active virions in at least one copy, again using a Ser-Gly linker. These studies suggest that with a suitable spacing linker and controlled levels of expression, it is likely that a wide range of protein or peptide substitutents can be fused with gpV at its C terminus and assembled as component subunits of the tail tube.
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PMID:Assembly of functional bacteriophage lambda virions incorporating C-terminal peptide or protein fusions with the major tail protein. 775 19

A protein modification method has been developed for the production of human big endothelin (ET)-1. Production of a large quantity of big ET-1 by the method described here is expected to facilitate future experiments such as X-ray crystallography and nuclear magnetic resonance studies, aimed at understanding the tertiary structure of big ET-1 and its dynamics. The plasmid pETB-50 used for the synthesis carries the gene for a fusion protein consisting of 34-amino acid (aa) residues of an N-terminal portion of beta-galactosidase and the 38-aa residues of big ET-1. The fusion protein ETB-50P contains an arginine residue in the big ET-1 portion at its second C-terminal site and three lysine residues including the C-terminal site in the beta-galactosidase portion, all of which are susceptible to trypsin. Tryptic digestion of the fusion protein quantitatively produced big ET-1 (1-37), which is depleted in the C-terminal serine. However, a treatment of the fusion protein with 1,2-cyclohexanedione prior to tryptic digestion gave full-length big ET-1 with N7, -N8-(1,2-dihydroxycyclohex-1,2- ylene)-arginine. This modification was reversed to the intact arginine residue when the modified big ET-1 was incubated in 0.5 M TRIS-HCl buffer, pH 8.0. Consequently, a combination of such a reversible protein modification and tryptic digestion gave 1.74 mg of recombinant big ET-1 from 2.01 of culture broth. The procedure described here may be applied to produce other arginine-containing peptides from fusion proteins.
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PMID:High-yield production of human big endothelin-1 by a combination of chemical modification and proteolysis of a fusion protein in Escherichia coli. 776 64

Cystathionine beta-synthase (CBS) purification from mammalian tissues is complicated by proteolysis and enzyme aggregation. To surmount these difficulties, we cloned human CBS cDNA in tandem with the beta-galactosidase sequence of the fusion vector, pAX5-, then expressed the fusion protein, beta-galactosidase/CBS, in transformed Escherichia coli cells. Proteolytic treatment of the ammonium sulfate fraction of bacterial lysates with endoproteinase Xa liberated CBS which could then be separated from its fusion partner by DEAE-cellulose chromatography. This nearly homogeneous enzyme preparation was purified 140-fold over the crude bacterial lysate with nearly 50% recovery, and its specific activity, 210 U/mg protein, was comparable to that purified from human liver. The purified enzyme contained pyridoxal 5'-phosphate and exhibited positive cooperativity toward S-adenosyl-L-methionine (Hill coefficient = 5.2; Kact = 34 microM). Km values of the cloned enzyme in the absence of AdoMet are 3.1 and 1.1 mM for serine and homocysteine, respectively. They are virtually identical to those from human hepatic CBS. A Soret absorbance band (lambda max = 428 nm) which shifted to 448 nm after reduction with sodium dithionite revealed the presence of heme in the enzyme. Expression of the fusion protein in E. coli with subsequent purification represents the first time this enzyme has been isolated in sufficient quantities for biophysical and biochemical investigation.
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PMID:Expression of human cystathionine beta-synthase in Escherichia coli: purification and characterization. 782 2

Individual membrane protein spanning sequences can promote protein export. To help define the sequence features necessary for this action, we identified mutations disrupting export mediated by the first spanning sequence (TM1) of the Escherichia coli serine chemoreceptor. Mutant spanning sequences were generated and characterized using beta-galactosidase and alkaline phosphatase gene fusions. The protein export function of TM1 was remarkably tolerant of single charged residues, and the introduction of pairs of charged amino acids was necessary to eliminate export. The results are accommodated by a model in which export requires a stretch of uncharged residues whose summed hydrophobicity exceeds a particular threshold value. This threshold approximates the minimum hydrophobicity required for cleavable signal sequence function. In addition, the threshold was near the minimum hydrophobicity observed for wild-type spanning sequences in a collection of topologically characterized membrane proteins.
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PMID:Mutations eliminating the protein export function of a membrane-spanning sequence. 796 39

The Escherichia coli serC-aroA operon encodes biosynthetic enzymes for unrelated amino acid biosynthetic pathways leading to the synthesis of serine and the aromatic amino acids. A serC-aroA-lac translational fusion was constructed in the vector pMC1403. Synthesis of beta-galactosidase from the serC-aroA-lac fusion was found to be enhanced in the presence of lactose as the sole carbon source. This enhancement was not observed in strains containing a cya or crp mutant. However, the exogenous addition of cAMP greatly increased the beta-galactosidase synthesis in the cya mutant strain. The serC-aroA mRNA content, analyzed by a dot blot assay, also appeared to increase in the serC+ aroA+ cells after the exogenous addition of cAMP. These findings unambiguously indicate that the expression of the serC-aroA operon is positively controlled by cAMP.
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PMID:Cyclic AMP-dependent expression of the Escherichia coli serC-aroA operon. 801 34

Avian infectious laryngotracheitis virus (ILTV), a herpesvirus, is a highly contagious pathogen that causes an upper respiratory tract infection in chickens. It is one of the major problems in the poultry industry worldwide. Current vaccines are not satisfactory due to the induction of latent infection. Here we describe a system for the construction of recombinant ILTV. A 4-kbp ILTV EcoRI DNA fragment was cloned into plasmid pUC13 and sequenced. Computer prediction revealed two potential open reading frames with 216 and 259 amino acid residues, respectively. The 259-residue polypeptide was serine-rich. The beta-galactosidase (beta-gal) gene of E. coli was cloned into the XhoI/Bg/II site of this DNA fragment, integrated into the ILTV genome via homologous recombination, expressed under the control of the immediate-early cytomegalovirus promoter, and caused the formation of blue plaques in the presence of X-gal. The insertion of a foreign gene into the ILTV genome and the successful expression of the incorporated gene demonstrated the potential for the construction of attenuated recombinant ILTV vaccines and the development of ILTV as vectors for polyvalent vaccines against avian upper respiratory tract infections.
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PMID:Construction of recombinant avian infectious laryngotracheitis virus expressing the beta-galactosidase gene and DNA sequencing of the insertion region. 803 Feb 40

The 67-kD elastin-binding protein (EBP) mediates cell adhesion to elastin and elastin fiber assembly, and it is similar, if not identical, to the 67-kD enzymatically inactive, alternatively spliced beta-galactosidase. The latter contains an elastin binding domain (S-GAL) homologous both to the aorta EBP and to NH2-terminal sequences of serine proteinases (Hinek, A., M. Rabinovitch, F. W. Keeley, and J. Callahan. 1993. J. Clin. Invest. 91:1198-1205). We now confirm the functional importance of this homology by showing that elastolytic activity of a representative serine elastase, porcine pancreatic elastase, was prevented by an antibody (anti-S-GAL) and by competing with purified EBP or S-GAL peptide. Immunohistochemistry of adult aorta indicates that the EBP exists as a permanent component of mature elastic fibers. This observation, together with the in vitro studies, suggests that the EBP could protect insoluble elastin from extracellular proteolysis and contribute to the extraordinary stability of this protein. Double immunolabeling of fetal lamb aorta with anti-S-GAL and antitropoelastin antibodies demonstrated, under light and electron microscopy, intracellular colocalization of the proteins in smooth muscle cells (SMC). Incubation of SMC with galactosugars to dissociate tropoelastin from EBP caused intracellular aggregation of tropoelastin. A tropoelastin/EBP complex was extracted from SMC lysates by coimmunoprecipitation and cross-linking, and its functional significance was addressed by showing that its dissociation by galactosugars caused degradation of tropoelastin by endogenous serine proteinase(s). This suggests that the EBP may also serve as a "companion" to intracellular tropoelastin, protecting this highly hydrophobic protein from self-aggregation and proteolytic degradation.
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PMID:67-kD elastin-binding protein is a protective "companion" of extracellular insoluble elastin and intracellular tropoelastin. 803 52

Serine proteinases of 42, 22 and 14 kDa were purified from the culture fluid of Streptomyces olivaceoviridis by FPLC. The first 14 amino acids at their N-termini were identical and coincide with the N-terminal amino acid sequence of 92-kDa chitinase, which was found to hydrolyse casein. The four proteins hydrolyse synthetic substrates at the carboxyl group of lysine and (more slowly) arginine. The 14-kDa endoproteinase releases only two fragments of 42 and 43 kDa from beta-galactosidase. When the pure 92-kDa chitinase was incubated at 37 degrees C in Tris.HCl buffer, it was cleaved into a 70-kDa chitinase and a 22-kDa proteinase which in its part is rapidly degraded to a 14-kDa proteinase.
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PMID:The 92-kDa chitinase from Streptomyces olivaceoviridis contains a lysine-C endoproteinase at its N-terminus. 805 94

The cyclic 3', 5' adenosine monophosphate (cAMP) binding pocket of the cAMP receptor protein (CRP) of Escherichia coli was mutagenized to substitute cysteine or glycine for serine 83; cysteine, glycine, isoleucine, or serine for threonine 127; and threonine or alanine for serine 128. Cells that expressed the binding pocket residue-substituted forms of CRP were characterized by measurements of beta-galactosidase activity. Purified wild-type and mutant CRP preparations were characterized by measurement of cAMP binding activity and by their capacity to support lacP activation in vitro. CRP structure was assessed by measurement of sensitivity to protease and DTNB-mediated subunit crosslinking. The results of this study show that cAMP interactions with serine 83, threonine 127 and serine 128 contribute to CRP activation and have little effect on cAMP binding. Amino acid substitutions that introduce hydrophobic amino acid side chain constituents at either position 127 or 128 decrease CRP discrimination of cAMP and cGMP. Finally, cAMP-induced CRP structural change(s) that occur in or near the CRP hinge region result from cAMP interaction with threonine 127; substitution of threonine 127 by cysteine, glycine, isoleucine, or serine produced forms of CRP that contained, independently of cAMP binding, structural changes similar to those of the wild-type CRP:cAMP complex.
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PMID:Mutagenesis of the cyclic AMP receptor protein of Escherichia coli: targeting positions 83, 127 and 128 of the cyclic nucleotide binding pocket. 806 99

Serine tRNA gene derivatives with altered anticodons were introduced to the temperature-sensitive serT42 mutant, whose tRNA(1Ser) shows a base substitution of A10 for wild type G10. When a low copy number vector-system was used, the growth and beta-galactosidase synthetic activity of the serT42 mutant were restored by complementation with the tRNA(5Ser) (T34) gene or the tRNA(1Ser) (G34) gene as well as the tRNA(1Ser) (wt) gene, but not with tRNA(5Ser) (wt), tRNA(1Ser) (A34) or tRNA(1Ser) (C34) genes at 42 degrees C. When multicopy vectors were used, the transformation even with tRNA(1Ser) (A10) gene restored the growth and beta-galactosidase synthetic activity at 42 degrees C. The tRNA(1Ser) (A10) showed no thermosensitivity in serine acceptor activity by in vitro assay. At 42 degrees C, the amount of tRNA(1Ser) (A10) in the serT42 mutant was almost the same as those in the wild type. The nucleotides in the tRNA(1Ser) (A10) were found to be fully modified like those in the wild type tRNA(1Ser). Both of the tRNAs transcribed from tRNA(5Ser) (T34) and tRNA(1Ser) (G34) genes showed serine acceptor activity. Modified nucleosides of these tRNAs were also analyzed.
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PMID:Suppression of the serT42 mutation with modified tRNA(1Ser) and tRNA(5Ser) genes. 806 26


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