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 gene coding for a Trichosanthes trypsin inhibitor analog (Ala-6-TTI) in which methionine at position 6 was replaced by alanine was synthesized chemically. The synthetic gene was cloned into plasmid pWR590-1 and expressed in Escherichia coli as a fusion protein composed of beta-galactosidase fragment of 590 amino acid residues and (Ala-6)-TTI, with methionine as a connecting residue. After cyanogen bromide cleavage and reduction of the fusion protein, followed by refolding with trypsin-Sepharose 4B as a matrix and affinity chromatography on the immobilized enzyme, the fully active (Ala-6)-TTI was obtained. The trypsin inhibitory activity and amino acid composition of the recombinant (Ala-6)-TTI were consistent with those of the natural one. The (Ala-6)-TTI gene was also cloned into the secretion expression vector, pVT102U/alpha, in Saccharomyces cerevisiae. In order to make the reading frame of the gene compatible with the vector, a nucleotide was inserted into the (Ala-6)-TTI gene via site-directed mutagenesis. The secreted (Ala-6)-TTI was purified and found to be correctly processed at the junction between the alpha-factor leader peptide and (Ala-6)-TTI downstream. Of the two expression systems, the latter is more advantageous in the high yield (greater than 2 mg/liter), easy purification and needlessness of disulfide refolding.
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PMID:Chemical synthesis, molecular cloning, and expression of the gene coding for the Trichosanthes trypsin inhibitor--a squash family inhibitor. 142 10

Synthetic DNA fragments containing the coding sequence for the serine proteinase inhibitor aprotinin, also known as bovine pancreatic trypsin inhibitor (BPTI) a Kunitz type inhibitor were fused to form a synthetic aprotinin gene by the method of Khorana and cloned into E. coli. The synthetic gene is characterized by the presence of certain restriction sites. These restriction sites are unique within the used cloning system. Therefore, a great number of modifications can be achieved easily by exchange of appropriate restriction fragments. Using this method the variant [Glu52]aprotinin was obtained starting from the aprotinin gene. Both genes were successfully expressed in E. coli as fusion proteins with beta-galactosidase using vector pUR 278. No translation products could be detected in four other expression system (pUR 108, pDR 540, pKK 223-3 and pUC 8). [Glu52]aprotinin was purified and renatured after cyanogen bromide cleavage of the fusion protein. This recombinant [Glu52]aprotinin shows exactly the same trypsin-inhibitory profile as natural aprotinin.
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PMID:Synthesis, cloning and expression of recombinant aprotinin. 244 12

Using specific rabbit antibody, one non-competitive (sandwich) and three variants of competitive enzyme-linked immunosorbent assays for basic pancreatic trypsin inhibitor (BPTI) were elaborated. For the assays peroxidase-antibody, two peroxidase-BPTI and beta-galactosidase-BPTI conjugates were applied. The best was competitive assay with peroxidase-BPTI conjugated by two step glutaraldehyde procedure. By this assay we were able to determine 1-10 ng BPTI/ml (0.007-0.07 KIU/ml) and the presence of human blood serum did not affect the assay of the inhibitor.
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PMID:Enzyme-linked immunosorbent assay of basic pancreatic trypsin inhibitor. 247 81

The distribution and localization of acid stable trypsin inhibitor (ASTI) in normal and malignant human tissues from various organs were examined using immunohistochemical techniques that used goat antibody raised against highly purified ASTI from human urine. Tissues were assessed as positive only when they were stained by both the biotin-avidin-peroxidase complex system and biotin-streptavidin-beta-galactosidase complex system, and the staining was abolished by absorption with purified ASTI. Under normal conditions, ASTI immunoreactivity was observed in only a few organs. Positive tissues for ASTI immunoreactivity included the kidney proximal tubules, glial cells of the cerebrum, fibrillar structures of the lamina propria of the stomach and colon, and bronchial epithelial cells. No ASTI immunoreactivity was observed in the cardiovascular system, reproductive system, or other tissues examined. As is not the case for normal tissues, ASTI immunoreactivity was found to be widely distributed in malignant tumors. Staining was observed in the extracellular space, i.e., in the stroma of the tumor and in connective tissues around the tumor invasion, whereas no ASTI immunoreactivity was detected in the malignant cells. Considering the identity of the first 36 NH2-terminal residues of ASTI purified from plasma or urine with a recently reported endothelial cell growth factor, the present findings suggest that ASTI could play an important role, not limited to its function as a protease inhibitor, in the invasive growth of malignant neoplasms.
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PMID:Distribution of acid stable trypsin inhibitor immunoreactivity in normal and malignant human tissues. 247 69

In the present study we have investigated the macromolecular nature of porcine oxyntic mucosal PGE2 binding sites and the involvement of specific functional groups in the binding interaction. Incubation of oxyntic mucosal membranes with DNAse or RNAse did not influence binding. Phospholipase A2 was strongly inhibitory while phospholipases C and D exerted variable effects. Trypsinization of the membranes also reduced binding and this reduction was prevented by addition of soybean trypsin inhibitor. Neuraminidase and beta-galactosidase treatments resulted in variable increases in binding activity. The increase in binding was due to an increase in binding affinity and/or binding site concentration. Protein modifying reagents acetic anhydride, N-ethylmaleimide and mercaptoethanol all reduced binding. These results suggest the importance of protein, lipid and carbohydrate components of the membrane in the binding interaction between PGE2 and its binding site. The ability of mercaptoethanol and N-ethylmaleimide to reduce binding suggest the involvement of both sulphydryl and disulphide groups in the PGE2 binding reaction.
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PMID:Effects of enzymes and protein modifying reagents on the binding of 3H-prostaglandin E2 to porcine oxyntic mucosa in vitro. 346 Jan 18

Four field strains of Lactobacillus plantarum (LS 4, 19, 21, 133) obtained from fufu (a semi-solid product obtained by boiling fermented cassava--Manihot esculenta Crantz) and a type strain DSM 2017 were grown on different carbon sources to induce galactosidase production. LS 21 produced the highest concentration of alpha- and beta-galactosidase with 0.28 mumol/l and 0.28 mumol/l respectively on lactose and galactose. Milk obtained from soybean seeds treated with the enzyme mixture for 24 h showed a 99, 98 and 96% reduction respectively in the raffinose, stachyose and sucrose content when compared with the dry soybean seed. Glucose and galactose which were not detected in the dry seeds became readily available after soaking in both enzyme mixture and distilled water. Although there was reduction in the nutritional composition of both milk samples, reduction of phytic acid and trypsin inhibitor is beneficial to the consumers. The result of the sensory evaluation showed that the milk prepared from enzyme-treated soybean seeds was rated better in terms of flavour, texture, appearance and palatability.
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PMID:Effect of bacterial galactosidase treatment on the nutritional status of soybean seeds and its milk derivative. 911 67

The stability of pKD1-based vectors was evaluated during the synthesis of intracellular and extracellular gene products in the yeast Kluyveromyces lactis. The Escherichia coli lacZ and MFalpha1 leader-BPTI (bovine pancreatic trypsin inhibitor) cassettes were placed under the control of the inducible K. lactis LAC4 promoter and inserted into the pKD1-based plasmids. To induce gene expression while maintaining inducer level, a gratuitous gal1-209 K. lactis strain was employed. Selective medium containing 5 g glucose/l and 0.5 g galactose (inducer)/l allowed optimum expression and secretion of heterologous products without a significant effect on the growth of the recombinant cells. During long-term sequential batch cultures (60 generations), plasmid instability was mainly the result of structural instability. The expression and secretion of BPTI resulted in greater structural instability relative to the intracellular beta-galactosidase. For both products, vectors carrying the pKD1 replication origin and the cis-acting stability locus (partial-pKD1 vectors) were more stable than vectors carrying the full pKD1 sequence (full-pKD1 vectors). However, after 55 generations, the beta-galactosidase and BPTI activities were still higher with the full-pKD1 vectors. This was due to the significantly higher initial beta-galactosidase and BPTI activities for the full-pKD1 vectors (approximately 85% and 47% higher, respectively) relative to the partial-pKDI vectors. Southern blots confirmed that these increases were due to the higher copy number of the vectors carrying the full pKD1 sequence. In contrast to our previously reported results for the secretion of invertase, full-pKD1 vectors were preferred for the expression/secretion of beta-galactosidase and BPTI for at least 55 generations. Due to their structural stability, partial-pKD1 vectors will be advantageous for very long cultivation times.
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PMID:Evaluation of pKD1-based plasmid systems for heterologous protein production in Kluyveromyces lactis. 1187 8

A gratuitous induction system in the yeast Kluyveromyces lactis was evaluated for the expression of intracellular and extracellular products during fed-batch culture. The Escherichia coli lacZ gene (beta-galactosidase; intracellular) and MFalpha1 leader-BPTI cassette (bovine pancreatic trypsin inhibitor; extracellular) were placed under the control of the inducible K. lactis LAC4 promotor, inserted into partial-pKD1 plasmids, and transformed into a ga1-209 K. lactis strain. To obtain a high level of production, culture conditions for growth and expression were initially evaluated in tube cultures. A selective medium containing 5 g/L glucose (as carbon source) and 0.5 g/L galactose (as inducer) demonstrated the maximum activity of both beta-galactosidase and secreted BPTI. This level of expression had no significant effect on the growth of the recombinant cells; growth rate dropped by approximately 11%, whereas final biomass concentrations remained the same. In shake-flask culture, biomass concentration, beta-galactosidase activity, and BPTI secreted activity were 4 g/L, 7664 U/g dry cell, and 0.32 mg/L, respectively. Fed-batch culture (with a high glucose concentration and a low galactose [inducer] concentration feed) resulted in a 6.5-fold increase in biomass, a 23-fold increase in beta-galactosidase activity, and a 3-fold increase in BPTI secreted activity. The results demonstrate the success of gratuitous induction during high-cell-density fed-batch culture of K. lactis. A very low concentration of galactose feed was sufficient for a high production level.
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PMID:Application of a gratuitous induction system in Kluyveromyces lactis for the expression of intracellular and secreted proteins during fed-batch culture. 1252 85