Recombinant Escherichia coli O9:H4 Beta-galactosidase (lacZ), partial
Description
Research and Development Insights
Mutational Analysis
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A study of 6,465 lacZ mutations identified 492 codons essential for β-galactosidase function, including residues near the catalytic site and subunit interfaces .
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Approximately 50% of lacZ codons contribute to structural or functional stability, highlighting the enzyme's complexity .
Recombinant Expression Systems
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Mammalian Cells: Recombinant lacZ constructs (e.g., pcDNA3.1-cyt.lacZ) have been transfected into HeLa cells, demonstrating cytoplasmic or nuclear localization depending on targeting sequences .
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Activity Metrics: Purified recombinant β-galactosidase exhibits >600 U/mg protein activity, measured via hydrolysis of p-nitrophenyl-β-D-galactopyranoside .
Applications in Biotechnology
Molecular Biology
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Reporter Gene: Widely used in α-complementation assays to screen recombinant DNA clones .
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Gene Delivery Studies: Serves as a marker to evaluate transfection efficiency in mammalian systems .
Industrial Uses
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Food Industry: Hydrolyzes lactose in dairy products for lactose-intolerant consumers .
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Enzyme Engineering: Truncated variants are optimized for stability and specificity in synthetic biology applications .
Comparative Analysis of Recombinant Forms
Critical Research Findings
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Domain Relevance: The TIM barrel domain (residues 20–627) is indispensable for substrate binding, while C-terminal regions aid tetramerization .
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Functional Redundancy: Even single mutations in 229 codons disrupt activity, confirming their roles in enzyme stability .
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Localization Studies: Nuclear-targeted recombinant β-gal (e.g., pcDNA3.1-nls.lacZ+) enables spatial tracking of gene expression in eukaryotic cells .
