Recombinant Enzyme Purification and Characterization

Extraction of genomic DNA, PCR with gene-specific primers (see Table S5 in the supplemental material), and cloning of genes into plasmids pET9a-USER-1 and pET9a-USER-2 were carried out as described previously (10 (link)). Expression of recombinant enzymes was carried out in ZYP-5052 autoinduction medium (40 (link)) supplemented with appropriate antibiotics, and cells were disrupted in a FastPrep-24 5G bead beater (MP Biomedicals) as described by Schultz-Johansen et al. (10 (link)). Recombinant, His-tagged enzymes were subsequently applied to a HisTrap FF column (GE Healthcare, Uppsala, Sweden) charged with 100 mM NiSO₄. After a washing step performed with 50 mM imidazole, the bound proteins were eluted with a linear gradient of imidazole ranging from 50 to 700 mM. Final protein concentration and purity were determined with a NanoDrop spectrophotometer (Thermo Scientific, Illkirch, France) and by SDS/PAGE, respectively. Extracts from E. coli cells treated with an empty vector were processed and analyzed in parallel as negative controls.

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Christiansen L., Pathiraja D., Bech P.K., Schultz-Johansen M., Hennessy R., Teze D., Choi I.G, & Stougaard P. (2020). A Multifunctional Polysaccharide Utilization Gene Cluster in Colwellia echini Encodes Enzymes for the Complete Degradation of κ-Carrageenan, ι-Carrageenan, and Hybrid β/κ-Carrageenan. mSphere, 5(1), e00792-19.

Publication 2020

FastPrep-24 5G bead beater HisTrap FF column NanoDrop spectrophotometer

Antibiotics Cells Cells extracts E coli Enzymes Genes Genomic Imidazole Plasmids Primers Protein Sds page Vector

Corresponding Organization :

Other organizations : University of Copenhagen, Korea University, Technical University of Denmark

Top 5 similar protocols

Variable analysis

independent variables

Expression of recombinant enzymes

dependent variables

Final protein concentration and purity

control variables

Genomic DNA extraction
PCR with gene-specific primers
Cloning of genes into plasmids pET9a-USER-1 and pET9a-USER-2
Cell disruption in a FastPrep-24 5G bead beater
Purification of recombinant, His-tagged enzymes using a HisTrap FF column
Washing step with 50 mM imidazole
Elution with a linear gradient of imidazole ranging from 50 to 700 mM

positive controls

Extracts from E. coli cells treated with an empty vector

negative controls

Extracts from E. coli cells treated with an empty vector

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