Wyszukiwarka
Identification and cloning of C. albicans SC5314 genes encoding L-methionine biosynthetic pathway enzymes.
Opis
Enzymes of fungal L-methionine biosynthetic pathway: homoserine O-acetyltransferase (Met2p), O-acetylhomoserine sulfhydrylase (Met15p) and cystathionine-γ-synthase (Str2p) could be exploited as molecular targets for antifungal chemotherapy. The goal of the study was to identify and clone genes encoding mentioned above enzymes. MET2, MET15 and STR2 genes were identified with the use of Candida Genome Database (http://www.candidagenome.org/). Amplifications of genes were carried out on C. albicans SC5314 genomic DNA with the use of polymerase chain reaction (PCR) and appropriate designed primers. PCR products that give satisfying molecular weight were selected and then cloned in a vector for its maintenance to produce the target protein. The constructed expression plasmids were designed in three different versions: encoding a native protein or recombinants with oligo-His tags added either at the C- or N-end. Introduction of fusion domains would facilitate future isolation of the gene products via metal-affinity chromatography. The additional oligo-His domains were introduced by PCR with the help of appropriate designed primers. A direct mutagenesis was carried out in a case of the plasmid containing MET2 gene because it contains a codon CTG that is translated differently in bacteria and yeast cells. In C. albicans it encodes L-Serine, whereas in E. coli cells L-Leucine.
This dataset contains the description of methodology, used primers sequences and three image files with the results of agarose gel electrophoresis of obtained PCR products, agarose gel electrophoresis of isolated plasmids DNA and restriction analysis.
The studies were financially supported from the project OPUS 20 financed by the National Science Centre (No. UMO-2020/39/B/NZ7/01519).
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- Licencja:
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Informacje szczegółowe
- Rok publikacji:
- 2022
- Data zatwierdzenia:
- 2022-01-18
- Język danych badawczych:
- angielski
- Dyscypliny:
-
- nauki chemiczne (Dziedzina nauk ścisłych i przyrodniczych)
- DOI:
- Identyfikator DOI 10.34808/zac2-tz70 otwiera się w nowej karcie
- Finansowanie:
- Weryfikacja:
- Politechnika Gdańska
Słowa kluczowe
- cloning
- methionine biosynthesis
- expression plasmids
- mutagenesis
- restriction digest analysis
- sequencing
Powiązane zasoby
- dane badawcze Overproduction of CaStr2p native and His-tag versions.
- dane badawcze Assay of Cystathionine-γ-synthase (CaStr2p) activity determination.
- dane badawcze Overproduction of homoserine O-acetyltransferase (CaMet2p) native and His-tag versions.
- dane badawcze Inserts amplification for knockout cassette construction
- dane badawcze Overproduction of CaMet15p native and His-tag versions.
- dane badawcze Antifungal activity of L-homoserine O-acetyltransferase (CaMet2p) inhibitors.
- dane badawcze L-Homoserine O-acetyltransferase (CaMet2p) activity and inhibition
- dane badawcze Plasmids pMET2M1 and pMET15M1 with the SAT1 Flipper knockout cassette construction
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