Isopropyl β d thiogalactopyranoside

The Escherichia coli JM109 competent cells, PrimeSTAR HS DNA polymerase, EmeraldAmp MAX PCR Master Mix, In-Fusion HD cloning kit, and NcoI were purchased from Takara Bio, Inc. (Shiga, Japan). The pQE-60 plasmid was obtained from Qiagen GmbH (Hilden, Germany). Poly(R)-3-hydroxybutyric acid, ( ±)3-hydroxybutyric acid (3HB) sodium salt and L-methionine sulfone were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Acetone, sulfuric acid, methanol, sodium D-pantothenate, β-alanine, ampicillin sodium salt (Ap), chloramphenicol (Cm), isopropyl β-D-thiogalactopyranoside (IPTG), ethanol for HPLC, and chloroform for HPLC were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Oligonucleotides for PCR and DNA sequencing were purchased from Eurofins Genomics K.K. (Tokyo, Japan). PIPES sesquisogium and D-camphor-10-sulfonic acid were purchased from Dojindo Laboratories Co., Ltd. (Kumamoto, Japan) and Nacalai Tesque, Inc. (Kyoto, Japan), respectively. Amicon Ultra-0.5 Centrifugal Filter Units (3 kDa) were obtained from Merck KGaA (Darmstadt, Germany). All other chemicals were of reagent grade.

The full length of SSA1 gene was amplified by PCR using the genomic DNA of C. albicans NBRC 1385 as template. The sequences of forward and reverse primers containing NdeI and BamHI recognition site (underlined) are 5′-GCCATATGTCTAAAGCTGTTGGTATT-3′ and 5′-GCGGATCCTTAATCAACTTCTTCAACAGTT-3′, respectively. AmpliTaq Gold DNA polymerase (Applied Biosystems; ThermoFisher, Waltham, MA, USA) and the following thermal protocol were used to amplify SSA1 gene: 95 °C for 10 min; 40 cycles of 95 °C for 1 min, 50 °C for 40 s, and 72 °C for 2 min; 72 °C for 10 min. The PCR product (1971 bp in size) was ligated into NdeI and BamHI-digested pET-15b vector (Novagen, Darmstadt, Germany) and transformed into Escherichia coli Rosetta (DE3) (Novagen). Protein expression was induced by 1 mM isopropyl-β-D-thiogalactopyranoside (Wako Pure Chemical Industries), and (His)6-Ssa1 fusion protein was purified using TALON^® Metal Affinity Resin (Takara, Tokyo, Japan), according to the manufacturer’s protocol. Lipopolysaccharide was removed from the purified protein using ProteoSpin Endotoxin Removal Mini Kit (Norgen Biotek Corp., Thorold, ON, Canada). The purified protein was confirmed by 8% SDS-PAGE. (His)6-tagged protein from the backbone pET-15b plasmid was purified by the similar method and used as control. The protein concentration was evaluated using Bio-Rad Protein Assay Dye reagent (BIO-RAD).

GST-fused EGF (GST-EGF) was constructed as previously described with some modifications (50 (link)). Human EGF DNA (accession: NP_001954) was synthesized by Eurofins Genomics. The ORF of EGF was amplified from the synthetic DNA using primers 5′-GGGGGATCCGGGATAAATGATAAAATGGAA-3′ and 5′-CCCGAATTCTTAACATCCTATAGAACCTAC-3′ and then inserted into the BamHI–XmaI site of pGEX6P-1 vector (Cytiva). Escherichia coli strain Rosetta-gami B(DE3) was transformed with this plasmid. Transformants were incubated at 20 °C for 24 h in the presence of 100 μM isopropyl-β-D-thiogalactopyranoside (FUJIFILM Wako Pure Chemical). GST-EGF expressed in soluble fractions was purified using glutathione-Sepharose 4B beads (Cytiva), and purified GST-EGF was dialyzed against PBS. This genetic recombination experiment was approved by the safety committee for genetic modification experiments of Niigata University (no.: SD01711).

Recombinant N-terminally His₆-tagged SWAN protein (rSWAN) was prepared using the E. coli-expression vector pQE30. Briefly, swan DNA encompassing the entire protein without the putative signal sequence and C-terminal sorting signal was amplified by PCR using the primers rSWANF 5′-gcggatccgaagaagcggtaagttcgtcg-3′ and rSWANR 5′-gccccgggggtcttcgggagtcccttctt-3′. The amplicon was digested with BamH I and Sma I, and cloned into pQE30 (Qiagen). All primers were designed using the genome sequence of S. sanguinis strain SK36 (Genbank accession number: CP000387.1) [26] (link). Hyperexpression of rSWAN was induced with 1 mM of isopropyl-β-D-thiogalactopyranoside (Wako) at 37°C for 5 h. The cells were lysed with 100 µg/ml of lysozyme (Wako) and intermittent sonication. Then, recombinant protein was purified from the lysates using a QIAexpress protein purification system (Qiagen) according to the manufacturer's instructions. Eluted protein was ultrafiltrated with Amicon Ultra 3K filter units (Millipore, MA, USA). The concentration was determined using a BCA protein assay kit (Pierce, IL, USA). Purity and integrity of the recombinant proteins were examined by SDS-PAGE and following Coomassie Brilliant Blue staining.