Phusion site directed mutagenesis protocol

The pcDNA 3.1-based genetic constructs for expression of FGFR1.GF variants with reintroduced mutations to alanines were obtained via gene synthesis (Gene Universal, Newark, DE, USA) or prepared using site-directed mutagenesis using Phusion™ Site-Directed Mutagenesis Protocol (Thermo Fisher Scientific) with pcDNA 3.1—FGFR1.GF as a template. The correctness of all genetic constructs was confirmed by DNA sequencing.

The endogenous AHA1 sequence was amplified using primers AHA1_BamHI and AHA1_XbaI containing cleavage sites for BamHI or XbaI at their 5′-ends. The PCR fragment was digested simultaneously with BamHI and XbaI subsequently cloned into the BamHI and XbaI sites of the expression vector pYES2/CT (Invitrogen). The wild-type AHA1 gene in the vector was mutagenized using the Phusion site-directed mutagenesis protocol (Thermo Scientific), to generate a series of clones with an amber stop codon at positions 59–66 (Table S1). The correct sequences of the corresponding plasmids (Table S3) were verified by sequencing.

Cloning via restriction sites was performed with PCR products amplified from S. solfataricus str. P2 genomic DNA. For co-expression of Sso TFEα (Sso0266) and Sso TFEβ-His (Sso 0944) a bicistronic expression construct was produced (p1076). A corresponding construct for co-expression of hRPC62 and hRPC39 (p1159) was created likewise. To generate a pRSF-1b-based vector for expression of C-terminally His-tagged Sso TFEβ, the gene was amplified from a pET21a+ expression vector including the vector-encoded His-tag and inserted into the pRSF-1b vector that does not encode a His-Tag otherwise. The resulting construct (p1077) was also used as backbone to generate the different Sso TFEβ truncation mutants. All plasmids generated by restriction enzyme-based cloning are listed in Supplementary file 1. Site-directed mutagenesis of TFEα and TFEβ was performed to yield single or double amino acid substitutions as listed in Supplementary file 2. For the deletion of nucleotides coding for residues 46–52 in Sso TFEα, a 5′-end phosporylated non-overlapping primer pair was used according to the Phusion Site-directed mutagenesis protocol (Thermo Scientific/Fisher, Loughborough, United Kingdom). All oligonucleotide sequences are listed in Supplementary file 3. The sequence of all constructs was verified.