Yeast extract peptone dextrose

Escherichia coli DH5α was purchased from TIANGEN (Beijing, China). P. pastoris X-33 and expression plasmid pPICZαA were conserved at −80 °C. EcoRІ, KpnІ, SacI, and universal DNA purification kits were purchased from TaKaRa (Beijing, China). Buffered glycerol-complex medium (BMGY), buffered methanol-complex medium (BMMY), and yeast extract peptone dextrose (YPD) were prepared based on the protocols provided by Invitrogen (Carlsbad, CA, USA).

Pichia pastoris GS115 and pPIC3.5K were purchased from Invitrogen Inc. (San Diego, CA, United States). E. coli DH5α and pET30a (+) were purchased from Novagen Inc. (Madison, WI, United States). The GS115-ZA-Pgp strain containing the sequence encoding P-glycoprotein was constructed previously (Esser et al., 2016 (link)). All the plasmids and strains were stored in our laboratory. Media including Luria-Bertani (LB), yeast extract peptone dextrose (YPD), minimal dextrose (MD), minimal methanol (MM), buffered glycerol complex (BMGY), buffered methanol complex (BMMY), basal salts medium (BSM), and Pichia trace metal (PTM) solution were prepared as per the Invitrogen’s multi-copy Pichia expression kit. Plasmid midi, PCR purification, and gel extraction kits were purchased from Qiagen Inc. (Chatsworth, CA, United States). The restriction enzymes, phusion high-fidelity DNA polymerase and T4 DNA ligase were obtained from New England Biolabs Inc. (Ipswich, MA, United States). p-Nitrophenyl-β-D-glucopyranoside (pNPG) was purchased through Sigma-Aldrich Inc. (St. Louis, MO, United States). Other chemicals were obtained from Thermo Fisher Scientific (Waltham, MA, United States). All other reagents used were of reagent grade. DNA primers synthesis and capillary DNA sequencing were performed by Center for Biologics Evaluation and Research (CBER of FDA, Silver Spring, MD, United States).

Rhizopus nigricans and Aspergillus flavus were obtained from the clinical microbiology laboratory and maintained on yeast extract peptone dextrose (Life Technologies, Grand Island, NY, USA) agar. The isolates were anonymous with respect to patient origin and did not derive from any of the autopsies. Both species were cultured in yeast extract peptone dextrose broth for 8–10 h at 37 °C, washed in tris-buffered saline, pH 7.4 and then heat-killed at 80 °C for 2 h in a water bath and washed in tris-buffered saline, pH 7.4 with 4% bovine serum albumin and 2 mM Ca⁺⁺. Fungi were fixed on microscope slides and incubated with human serum (the source of SAP) or tris-buffered saline, pH 7.4, with 4% bovine serum albumin and 2 mM Ca⁺⁺ for 2 h, washed with buffer and incubated with SAP antibody (Biocare Medical) for 1 h. The slides were washed again with the same buffer and fungi incubated with 1/500 Alexa Fluor 555 goat anti-rabbit IgG (Life Technologies) in tris-buffered saline with 4% bovine serum albumin and 2 mM Ca⁺⁺ for 1 h, washed and observed with a DeltaVision deconvolution microscope (GE Healthcare, Issaquah, WA, USA). All incubations were performed at 26 °C. Both antibody preparations were pre-absorbed against the respective heat-killed fungi overnight at 26 °C.