Purification of Mutant ADI Protein

ADI (amino acids 1 to 411, D277A) previously cloned into the expression vector pET151/D-TOPO [13 (link)], was expressed in E. coli BL21 Star (DE3) cells and purified by immobilised metal ion affinity chromatography (IMAC) as previously reported [12 (link)]. Endotoxins were removed by washing the IMAC-bound protein with Triton X-114 [20 (link)]. Final protein concentration was determined using a Direct Detect infrared spectrometer (Millipore, Burlington, MA, USA) and endotoxin levels were measured using the Pierce Limulus amebocyte lysate (LAL) chromogenic endotoxin quantitation kit (Thermo Fisher Scientific, Waltham, MA, USA).

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Rivera-Hernandez T., Carnathan D.G., Richter J., Marchant P., Cork A.J., Elangovan G., Henningham A., Cole J.N., Choudhury B., Moyle P.M., Toth I., Batzloff M.R., Good M.F., Agarwal P., Kapoor N., Nizet V., Silvestri G, & Walker M.J. (2024). Efficacy of Alum-Adjuvanted Peptide and Carbohydrate Conjugate Vaccine Candidates against Group A Streptococcus Pharyngeal Infection in a Non-Human Primate Model. Vaccines, 12(4), 382.

Publication 2024

Direct Detect infrared spectrometer Pierce Limulus amebocyte lysate (LAL) chromogenic endotoxin quantitation kit

Corresponding Organization :

Other organizations : Hospital de Especialidades, University of Queensland, Centro Medico Nacional Siglo XXI, Mexican Social Security Institute, Emory University, University of California, San Diego, Griffith University

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Variable analysis

independent variables

Expression of ADI (amino acids 1 to 411, D277A) in E. coli BL21 Star (DE3) cells

dependent variables

Protein concentration of the final purified sample
Endotoxin levels in the final purified sample

control variables

Purification of the expressed protein by immobilised metal ion affinity chromatography (IMAC)
Removal of endotoxins by washing the IMAC-bound protein with Triton X-114

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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