Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.3.8 (phytase)
 1,997 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Gene synthesis technologies provide a powerful tool for increasing protein expression through codon optimization and gene modification. Here we describe an improved PCR-based gene synthesis technology, which is accurate, simple and cheap. The improved PCR-based gene synthesis (IPS) method consists of two steps. The first one is the synthesis of 300-400bp fragments by PCR reaction with Pfu DNA polymerase from 60-mer and 30-mer oligonucleotides with a 15bp overlap. The second one is assembling of fragments from the first step into the full-length gene by PCR reaction. Using this approach, we have successfully synthesized a modified phytase gene with 1256bp in length with optimal codons for expression in Pichia pastoris. P. pastoris strain that expressed the modified phytase gene (phyA-mod) showed a 50% increase in phytase activity level. In addition, we propose an inexpensive method for error correction, based on overlap-extension PCR (OE-PCR).
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PMID:Improved PCR-based gene synthesis method and its application to the Citrobacter freundii phytase gene codon modification. 2022 18

Escherichia coli is a common host for recombinant protein production in which production titers are highly dependent on the employed expression system. Promoters are thereby a key element to control gene expression levels. In this study, a novel PLICable promoter toolbox was developed which enables in a single cloning step and after a screening experiment to identify out of ten IPTG-inducible promoters (T7, A3, lpp, tac, pac, Sp6, lac, npr, trc and syn) the most suitable one for high level protein production. The target gene is cloned under the control of different promoters in a single and efficient cloning step using the ligase-free cloning method PLICing (phosphorothioate-based ligase-independent gene cloning). The promoter toolbox was firstly validated using three well producible proteins (a cellulase from a metagenome library, a phytase from Yersinia mollaretii and an alcohol dehydrogenase from Pseudomonas putida) and then applied to two enzymes (3D1 DNA polymerase and glutamate dehydrogenase mutant) which are poorly produced in E. coli. By applying our PLICable pET-promoter toolbox, the authors were able to increase production by two-fold for 3D1 DNA polymerase (lac promoter) and 29-fold for glutamate dehydrogenase mutant H52Y (trc promoter).
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PMID:Screening through the PLICable promoter toolbox enhances protein production in Escherichia coli. 2775 30