pMCSG7-PP-CX

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	The PCR products were purified, T4 polymerase treated [11], cloned into the pMCSG7 vector series according to the LIC procedure [16], and transformed into the E.coli BL21 (DE3)-Goldstrain.

Protocol tips
The PCR products were purified, T4 polymerase treated [11], cloned into the pMCSG7 vector series according to the LIC procedure [16], and transformed into the E.coli BL21 (DE3)-Goldstrain.

Publication protocol

The selected genes were amplified by PCR from the genomic DNA with KOD Hot Start DNA polymerase. The PCR products were purified, T4 polymerase treated [11], cloned into the pMCSG7 vector series according to the LIC procedure [16], and transformed into the E.coli BL21 (DE3)-Goldstrain (Stratagene, Santa Clara, CA, USA) (which harbors an extra plasmid (pMgk) encoding one rare tRNA (corresponding to rare Arg codons, AGG and AGA)). These vectors are available from the PSI-MR material repository [8].The’Eps-RBS-fusion-strategy’ creates an internal Eps-RBS site by utilizing the forward 5′-GTTTAACTTTAAGAAGGAGATATACAT-3′ and reverse 5′-TCCTTCTTAAAGTTAAACACCATTCTA-3′ primers in the first step of the PCR reaction. The expression vectors were confirmed by DNA sequencing. The PP-CX expression and solubility is assessed in a small-scale (1 mL). A single colony is picked and grown overnight in 1 mL LB media with 150 μg/mL of ampicillin and 30 μg/Ml of kanamycin at 37 °C. The following morning, 120 μL of overnight culture is transferred to 48-well culture plates containing 4 mL of “pink” media. After reaching OD600 = 1 (~3 h of incubation at 37 °C, 270 rpm) the 48-well plates are cooled down to 18 °C and IPTG is added to a final concentration of 0.5 mM. The culture is grown for an additional 16–20 h at 18 °C, 270 rpm. The 96-well plates are then spun down, the supernatant is discarded, and the pellets are resuspended in 100 μL of buffer A [lysis buffer, 20 mM imidazole, 10 mM β-mercaptoethanol (β-ME)]. The plates are flash frozen in liquid nitrogen for about 10 min followed by batch sonication. After sonication and thawing, 20 μL of lysozyme and 20 μL of bensonaze (Novagen) are added to 15 mL of buffer A, then 150 μL of this solution is added to each well. Plates are then incubated at 26 °C, 200 rpm. After 90 min of incubation, disrupted cells are transferred from 48- to 96-well plates. At this point, expression samples are collected. The 96-well plates are spun at 3200 × g for 60 min. The supernatant is mixed with Ni Sepharose and applied onto a1 μm 96-well filter plate. Any unbound sample is washed out by 3 spinning/washing cycles of 250 μL at 50 × g. In the next step, 100 μL of elution buffer (500 mM imidazole in lysis buffer, TEV protease 0.2 mg/mL) is added to each well. The filter plate is spun down the following day and collected samples are analyzed by SDS-PAGE for the presence of the PP-CX. Only the targets with confirmed high-level expression and good solubility are moved into the large-scale fermentation.

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