Nature research paper: Biosynthetic potential of the global ocean microbiome
Chemically competentBL21 was introduced into pCDFDuet-1-EmbA and pCDFDuet-1-EmbM. The same conditions were used for expression of both N-terminally His-tagged proteins. Overnight cultures were prepared from single colonies and used to inoculate TB medium in 1 l baffled Erlenmeyer flasks supplemented with spectinomycin . Cells were grown at 37 °C, 200 rpm, until an optical density at 600 nm of around 1.0, cooled on an ice bath for 20 min and induced with a final concentration of 0.5 mM IPTG.
Modified EmbA was proteolysed with trypsin for MS analysis and with LahT150 for MS analysis and product isolation . Trypsin cleavage was performed by diluting the reaction mixture with 2× trypsin buffer , adding 1:20 trypsin:EmbA and incubating overnight at 37 °C. LahT150 cleavage was performed by adding LahT150 at a 1:10 ratio to EmbA, and incubating at room temperature overnight for small-scale reactions, or for 24 h, for reactions larger than 1 ml.
Large-scale enzymatic reactions were purified using solid-phase extraction with a Phenomenex Strata C18-E reverse phase column . The sorbent was first washed with 24 ml MeOH and equilibrated with 24 ml HO . The peptide products were eluted with 24 ml of 1:1 MeCN:HO and 12 ml MeCN . Elution fractions were pooled, dried on a Genevac concentrator and stored at −20 °C.
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