Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: DrugBank:EXPT01586 (G418)
 2,237 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Coding sequence cartridges for aminoglycoside phosphotransferase (APT) were isolated from bacterial transposon Tn903. When incorporated into a heterologous gene construction utilising the PGK1 promoter and terminator, the heterologous APT gene provided a G418-resistance determinant that functioned efficiently as a dominant marker for yeast in both multiple- and single-copy. Transformant colonies on selective medium appeared rapidly, within 36-48 h, and growth rate of the transformed cells was normal. A simple and highly sensitive radiolabelling assay for APT enzyme activity was developed for use with crude cell protein extracts. Enzyme activity units were equated to the amount of APT protein present in the cells, and the APT protein was shown to be stable in yeast. Heterologous APT expression was 130-fold reduced compared with homologous PGK1. This resulted from an estimated two-fold decrease in mRNA level and a 65-fold decrease in translation efficiency. The latter was unaffected by AUG sequence context change, but corresponded with a high frequency of minor codons in the APT-coding sequence. APT can be used as a semi-quantitative reporter of gene expression, whose useful features are in vivo detection via the G418-resistance phenotype and powerful cell-free assay.
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PMID:G418-resistance as a dominant marker and reporter for gene expression in Saccharomyces cerevisiae. 217 44

The bglS gene encoding endo-l,3-1,4-beta-glucanase from Bacillus subtilis was cloned and sequenced in this study. The bglS expression cassette, including PGK1 promoter, bglS gene fused to the signal sequence of the yeast mating pheromone alpha-factor (MFalpha1(S)), and ADH1 terminator with G418-resistance as the selected marker, was constructed. Then one of the PEP4 allele of Saccharomyces cerevisiae WZ65 strain was replaced by bglS expression cassette using chromosomal integration of polymerase chain reaction (PCR)-mediated homologous recombination, and the bglS gene was expressed simultaneously. The recombinant strain S. cerevisiae (SC-betaG) was preliminarily screened by the clearing hydrolysis zone formed after the barley beta-glucan was hydrolyzed in the plate and no proteinase A (PrA) activity was measured in fermenting liquor. The results of PCR analysis of genome DNA showed that one of the PEP4 allele had been replaced and bglS gene had been inserted into the locus of PEP4 gene in recombinant strains. Different endo-l,3-1,4-beta-glucanase assay methods showed that the recombinant strain SC-betaG had high endo-l,3-1,4-beta-glucanase expression level with the maximum of 69.3 U/(h.ml) after 60 h of incubation. Meanwhile, the Congo Red method was suitable for the determination of endo-l,3-1,4-beta-glucanase activity during the actual brewing process. The current research implies that the constructed yeast strain could be utilized to improve the industrial brewing property of beer.
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PMID:Construction of recombinant industrial Saccharomyces cerevisiae strain with bglS gene insertion into PEP4 locus by homologous recombination. 1860 Jul 82

In this study, we constructed a novel and simple yeast surface display system with a single expression vector. The newly established system uses a bidirectional expression vector carrying the AGA1 gene driven by the PGK1 promoter in one direction and the AGA2-expression cassette driven by the TEF1 promoter in the reverse direction, and uses the geneticin, a G418-resistant gene, as the selection marker for transformants. Because all the display elements are put into one expression vector, the new system is much simpler to use, and there is no need for any genetic modification of the host strains; therefore, the new system can be used in wild type as well as laboratory strains of Saccharomyces cerevisiae. The display efficiency of heterologous proteins using the new system has been confirmed by displaying enhanced green fluorescent protein and Eimeria tenella (a chicken protozoan parasite) microneme protein2 (EtMic2) on several S. cerevisiae strains. We also tested the new system with an aga2 mutant strain of S. cerevisiae. The results indicate that the native expressed Aga2 protein has no effect on the display efficiency of heterologous proteins.
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PMID:Display of heterologous proteins on the Saccharomyces cerevisiae surface display system using a single constitutive expression vector. 2485 Dec 54