Computational protocol: Biochemical properties and yields of diverse bacterial laccase-like multicopper oxidases expressed in Escherichia coli

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Protocol publication

[…] Genes encoding putative LMCOs were identified in bacterial genomes by Protein BLAST searches (http://blast.ncbi.nlm.nih.gov/Blast.cgi) using sequences of the following bacterial proteins with confirmed ABTS oxidising activity as search templates: B. subtilis CotA (NCBI reference sequence NP_388511.1), S. coelicolor A3 copper oxidase (NCBI reference sequence NP_630785.1) and G. forsetii blue copper oxidase (NCBI reference sequence YP_861212.1, this study). The presence of putative N-terminal signal sequences was analysed with the Signal P 3.0-HMM program using appropriate networks for the analysed organisms (http://www.cbs.dtu.dk/services/SignalP/). Alignments of amino acid sequences and calculations of amino acid sequence similarities were carried out with the MegAlignTM software (LASERGENE, Madison, USA) using the ClustalW algorithm.Plasmids used in this study for expression of bacterial LMCOs are listed in . Primer sequences and details of plasmid construction are described in the Supplementary Material. Genomic and plasmid DNA was extracted with commercial kits according to the instructions of the suppliers. Gene sequences, cloning sites and upstream and downstream regions at the 5′ and 3′ ends of LMCO-encoding inserts were controlled by sequencing. The nucleotide sequences of all genes cloned from genomic DNA matched the corresponding database sequences to 100%. E. coli BL21(DE3) was used as host strain for pET derived plasmids. E. coli JM109 (overexpressing lacIq from the genome) was used as host strain for pQE-60 derived plasmids. In order to test the effect of additional copies of rare tRNAs, E. coli Rosetta 2 (DE3) competent cells (Novagen) harboring pRARE2 were transformed with pGoL3, pSiL, pMtraL, pBuL, pLOM10, pBoL and pBaL. JM109 competent cells were transformed with pRARE2, previously isolated from Rosetta 2 cells, made competent and then transformed with pSpL3. […]

Pipeline specifications

Software tools DELTA-BLAST, SignalP, Clustal W
Application Amino acid sequence alignment
Organisms Escherichia coli, Bacillus subtilis, Bacillus clausii, Bacillus coagulans
Diseases Bacterial Infections
Chemicals Copper, Oxygen