L1S protein was expressed in E. coli with a pTrcHis (Invitrogen) construct and purified as previously described (23 (link)). Briefly, log phase bacteria were incubated 8 h with IPTG to induce protein expression. Following cell lysis and DNAse treatment, lysates with centrifuges to pellet cellular debris. Supernatants were filtered in a 0.22 μm syringe filter and loaded onto and ATKA FPLC for nickel (Ni) column affinity purification of the 6 x His tagged protein. Aliquots of fractions eluted from the column were evaluated using SDS-PAGE and Coomassie staining. In some experiments, polymixin B columns were used to further purify L1S protein. Fractions containing L1S protein were concentrated using Millipore 10k KD columns and protein concentrations were determined using BCA.
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Affinity Purification of Recombinant L1S Protein
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Corresponding Organization : National Jewish Health
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Variable analysis
independent variables
- Expression of L1S protein in E. coli with a pTrcHis (Invitrogen) construct
- Incubation duration with IPTG to induce protein expression (8 hours)
- Purification of L1S protein
- Evaluation of fractions eluted from the column using SDS-PAGE and Coomassie staining
- Concentration of fractions containing L1S protein using Millipore 10k KD columns
- Determination of protein concentrations using BCA
- Cell lysis and DNAse treatment conditions
- Centrifugation conditions to pellet cellular debris
- Filtration of supernatants through a 0.22 μm syringe filter
- Nickel (Ni) column affinity purification of the 6 x His tagged protein
- Use of polymixin B columns to further purify L1S protein (in some experiments)
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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