Site-Directed Mutagenesis of AlbC Variants

Genes coding the six new single point variants of AlbC (R80A, R102A, N159A, R160A, D163A, D205A) were obtained via polymerase chain reaction (PCR) mutagenesis of the plasmid pQE60-AlbC encoding C-terminal His₆-tagged AlbC (5 (link)) according to the QuikChange^TM site-directed mutagenesis method (Stratagene). Sequences were verified by DNA sequencing. Plasmids encoding the other AlbC variants used in this study (R91A, K94A, R98A, R98A-R99A) were constructed previously (5 (link)).
The C-terminal His₆-tagged proteins were produced and purified as described previously (2 (link),5 (link)). Purified proteins were quantified by UV spectrophotometry. Protein molecular weights were verified by electrospray ionisation mass spectrometry (Esquire HCT ion trap mass spectrometer (Bruker Daltonik, GmbH)).

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Moutiez M., Seguin J., Fonvielle M., Belin P., Jacques I.B., Favry E., Arthur M, & Gondry M. (2014). Specificity determinants for the two tRNA substrates of the cyclodipeptide synthase AlbC from Streptomyces noursei. Nucleic Acids Research, 42(11), 7247-7258.

Publication 2014

Esquire HCT ion trap mass spectrometer

Electrospray ionisation mass spectrometry Genes Mutagenesis Plasmids Polymerase chain reaction Protein Site directed mutagenesis Spectrophotometry

Corresponding Organization :

Other organizations : Commissariat à l'Énergie Atomique et aux Énergies Alternatives, Université Paris Cité, Sorbonne Université, Centre de Recherche des Cordeliers, Inserm, Délégation Paris 5, Sorbonne Paris Cité

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Variable analysis

independent variables

Six new single point variants of AlbC (R80A, R102A, N159A, R160A, D163A, D205A)

dependent variables

Protein molecular weights verified by electrospray ionisation mass spectrometry

control variables

C-terminal His6-tagged AlbC (as encoded by plasmid pQE60-AlbC)
Other AlbC variants (R91A, K94A, R98A, R98A-R99A) constructed previously

controls

Positive control: C-terminal His6-tagged AlbC (as encoded by plasmid pQE60-AlbC)
Negative control: Not explicitly mentioned

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