Part:BBa_K2238001:Design
Glucose oxidase (A. niger) Quadruple Mutant
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 36
Illegal BamHI site found at 346 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 370
Illegal BsaI.rc site found at 1588
Design Notes
The amino acid sequence was converted to the E. coli optimized DNA coding sequence (IDT https://www.idtdna.com/CodonOpt). We added the coding sequence for an N-terminal His-tag and TEV protease for convenient protein isolation. This leaves a small glycine scar on the N-terminal end of the protein following enzymatic cleavage. There is a short 4-glycine-Serine tag followed by a SacI site prior to the stop codon. This was added for flexibility with potentially adding peptide and protein fusions.
The following mutations were made with consideration for codon optimization in E. coli. T56V, T132S, H469C, C543V
Source
Aspergillus niger Gene Bank: AAA32695.1 (E.C 1.1.3.4) Wild type sequence. Mutations include: T56V, T132S, H469C, C543V
References
J. Todd Holland, Jason C. Harper, Patricia L. Dolan, Monica M. Manginell, Dulce C. Arango, Julia A. Rawlings, Christopher A. Apblett, Susan M. Brozik. Rational Redesign of Glucose Oxidase for Improved Catalytic Function and Stability. PLoS ONE, 2012 vol. 7 (6) pp. e37924-10
J. Todd Holland, Carolin Lau, Susan Brozik, Plamen Atanassov, and Scott Banta. Engineering of Glucose Oxidase for Direct Electron Transfer via Site-Specific Gold Nanoparticle Conjugation. J. Am. Chem. Soc., 2011 vol. 133(48) pp. 19262-19265