E coli one shot bl21 de3

The primers aat_Fw (5′-ATGAGTGATAAAGTTAACGC-3′) and aat_Rv (5′-TGCTCGTTTAGCTTTGAGG-3′) were designed to amplify the complete aminotransferase-coding gene presented by the locus GBO44_RS03485. The primer aat_Rv did not contain the stop codon for cloning reasons. The amplification reactions were performed using the AmpliTaq Gold DNA Polymerase (Thermo Fisher Scientific) and applying the manufacturer’s protocol. Five nanograms of genomic DNA were used in 25 µL reaction volume. The amplification occurred in a thermal cycler using the following program: 10 min at 95 °C; 30 cycles of 15 s at 95 °C, 30 s at 45 °C, and 1 min at 72 °C; 5 min at 72 °C. The reaction products were analyzed using agarose gel electrophoresis.
The DNA fragment obtained from strain FAM18098 was cloned into the pEXP5-CT/TOPO expression vector (Thermo Fisher Scientific) applying the manufacturer’s protocol. The vector contains a nucleotide sequence encoding a poly-histidine tag that is added to the 3′-end of the DNA fragment. The recombinant vector was first transformed into E. coli TOP10. The plasmids extracted from transformants were verified using Sanger sequencing at Microsynth (Balgach, Switzerland). A plasmid containing the DNA fragment in the proper orientation was named pEXP5-CT/aat and transformed into E. coli One Shot BL21(DE3) (Thermo Fisher Scientific) for protein production.

For DYRK1A protein expression, One-shot E.coli BL21(DE3) (Invitrogen) was transformed with DYRK1A-pNIC28 vector (Addgene, Cat# 38913). The bacteria were grown at 37 °C in 2xYT medium containing 100 μg/ml Carbenicillin until A600nm reached ~0.6, cooled down to room temperature, followed by 0.5 mM IPTG induction of protein expression at room temperature for 16 hours. The DYRK1A protein was purified using the standard protocol for Ni-NTA purification under native conditions as described in “The QIAexpressionist Handbook”.
For DYRK1A in vitro kinase assay, immunoprecipitated human or chimeric, wild-type or T34A, MTHFR was incubated with 3 μg of DYRK1A in a kinase assay buffer (50 mM HEPES, pH7.3, 10 mM MgCl₂, 10 mM MnCl₂, 1 mM DTT, and 100 μM ATP) at 30 °C for 90 min. The reaction was stopped by adding Invitrogen LDS 4X sample buffer.