The cloning, DNA sequence, and overexpression of the gene araE coding for arabinose-proton symport in Escherichia coli K12
| Autor: | M C Jones-Mortimer, Peter J. F. Henderson, Martin C. J. Maiden |
|---|---|
| Rok vydání: | 1988 |
| Předmět: |
Expression vector
food and beverages Cell Biology biochemical phenomena metabolism and nutrition Biology Lambda phage medicine.disease_cause biology.organism_classification Biochemistry Molecular biology PBR322 carbohydrates (lipids) chemistry.chemical_compound Plasmid Restriction map chemistry medicine Molecular Biology Escherichia coli Peptide sequence DNA |
| Zdroj: | Journal of Biological Chemistry. 263:8003-8010 |
| ISSN: | 0021-9258 |
| DOI: | 10.1016/s0021-9258(18)68433-9 |
| Popis: | A lambda placMu1 insertion was made into araE, the gene for arabinose-proton symport in Escherichia coli. A phage containing an araE'-'lacZ fusion was recovered from the lysogen and its restriction map compared with that of the 61-min region of the E. coli genome to establish the gene order thyA araE orf lysR lysA galR; araE was transcribed toward orf. A 4.8-kilobase SalI-EcoRI DNA fragment containing araE was subcloned from the phage lambda d(lysA+ galR+ araE+) into the plasmid vector pBR322. From this plasmid a 2.8-kilobase HincII-PvuII DNA fragment including araE was sequenced and also subcloned into the expression vector pAD284. The araE gene was 1416-base pairs long, encoding a hydrophobic protein of 472 amino acids with a calculated Mr of 51,683. The amino acid sequence was homologous with the xylose-proton symporter of E. coli and the glucose transporters from a human hepatoma HepG2 cell line, human erythrocytes, and rat brain. The overexpressed araE gene product was identified in Coomassie-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels of cell membranes as a protein of apparent Mr 35,000 +/- 1,150. Arabinose protected this protein against reaction with N-ethylmaleimide. |
| Databáze: | OpenAIRE |
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