Proteins were purified as 6× His-tag fusion at the N terminus. E. coli BL21 (DE3) with plasmids were inoculated into LB medium with 200 mg l−1 ampicillin. Cells were induced with 0.5 mM IPTG, and the proteins were expressed for 24 h at 30 °C while shaking at 250 r.p.m. BM3 cytochrome P450 was extracted with the BugBuster Plus Lysonase Kit (EMD Millipore) and purified using the HisPur Ni-NTA Superflow Purification System (Thermo Fisher) according to the manufacturer’s instructions. Others proteins were purified using His-Spin Protein Miniprep kit (Zymo Research Corporation). The purified proteins were quantified by Bradford assay.
Bugbuster plus lysonase kit
Manufactured by Merck Group
The BugBuster Plus Lysonase Kit is a laboratory product designed for the efficient lysis and extraction of proteins from bacterial cells. The kit contains reagents and enzymes necessary for the effective disruption of bacterial cell walls and membranes, allowing for the release and purification of targeted proteins. The core function of this kit is to provide a streamlined solution for the extraction and preparation of proteins from bacterial samples for further analysis and study.
Automatically generated - may contain errors
2 protocols using bugbuster plus lysonase kit
1
BM3 Cytochrome P450 Protein Purification
2
Purification of VHH Fusion Proteins
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The bacteria were lysed using BugBuster Plus Lysonase kit (EMD Millipore; catalog no. 71370). His6-tagged thioredoxin-VHH fusion protein in the supernatant was purified using Ni-nitrilotriacetic acid (Ni-NTA) agarose (Thermo Fisher Scientific) and eluted with 250 mM imidazole. Purified VHH was dialyzed overnight in a 7-kDa-cutoff Slide-A-Lyzer dialysis cassette (Thermo Fisher Scientific) against 4 L PBS with at least one buffer change. Dialyzed protein was sterilized using a Whatman Puradisc 0.2-μm filter (Thermo Fisher Scientific) and analyzed for purity and concentration.
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