Dithiothreitol (dtt)

Proteins were extracted by 0.1 M Tris–HCl (pH 8.0) and 8 M urea with protein phosphatase inhibitor cocktails (Bioman, Taiwan) and protease inhibitors (BioShop, Canada). Protein concentrations were quantified using a Bio-Rad Protein Assay (Bio-Rad, Munich, Germany). The protein extracts of each sample was reduced with 10 mM dithiothreitol (Wako, Japan) for 45 min at room temperature, and then carbamidomethylated with 50 mM iodoacetamide (Sigma, USA) for 45 min at room temperature in darkness. Alkylated proteins were diluted four times with 50 mM triethylammonium bicarbonate (TEAB) and digested with endopeptidase Lys-C (1:100 w/w) (Wako) for 2 h. Subsequently, sequencing-grade modified trypsin (1:100 w/w) (Promega, Germany) was added for 16–18 h at room temperature. The digested peptides were acidified to a pH < 3 with trifluoroacetic acid (TFA). Acidified peptides were desalted using StageTips with SDB-XC Empore disc membranes (SDB-XC StageTip) (3 M, Germany) [66 (link)], and eluted in a buffer containing 0.1 % TFA and 80 % acetonitrile.

Transformation of K. marxianus was performed as previously described [30 (link)]. Briefly, yeast cells (RAK3908) were cultured in 30 ml of YPD medium in a 250 ml baffled flask and shaken at 150 rpm for 24 h at 30°C. The cells were collected by centrifugation and suspended in 900 μl of transformation buffer (TFB), prepared by mixing 20 ml of 60% polyethylene glycol 3350 (Sigma-Aldrich, Tokyo, Japan), 3 ml of 1 M dithiothreitol (Wako, Osaka, Japan), 1.5 ml of 4 M lithium acetate (Kishida Chemical, Osaka, Japan), and 5.5 ml of sterilized water. Next, the cells were centrifuged and resuspended in fresh 600 μl of TFB. Then, 50 μl of the cell suspension was mixed with the amplified DNA fragment (~70 ng) and incubated at 42°C for 30 min. The cell suspension was spread on a synthetic drop-out medium plate and incubated at 28-30°C for 2–3 d.

We purchased the therapeutic mAb immunoglobulin (IgG) gamma 1 drugs adalimumab and rituximab from Eisai Co., Ltd. (Tokyo, Japan) and Chugai Pharmaceutical Co., Ltd. (Tokyo, Japan), respectively. In addition, we purchased Tris-HCl buffer (pH 8.0), 10% trifluoroacetic acid, 0.1% formic acid, acetonitrile 0.1% formic acid and 7 K Dialysis Casettes from Thermo Fisher Scientific (San Jose, CA, USA). We obtained 8 M guanidine hydrochloride from Sigma-Aldrich (St. Louis, MO), dithiothreitol, iodoacetamide, sodium acetate, 2-morpholinoethanesulfonic acid, 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid and deuterium oxide from FUJIFILM Wako Pure Chemical Co., Ltd. (Tokyo, Japan), and MicroSpin G-25 columns from GE Healthcare (Chicago, IL). We purchased trypsin from Promega Co. (Madison, WI); angiotensin II from Peptide Institute, Inc. (Osaka, Japan); and ¹⁸O-water, acetonitrile (LC-MS grade), and ordinary water (LC-MS grade) from Merck (Darmstadt, HE).

Rat liver microsomes were prepared by the method of Lee et al. [12 (link)]. Male Sprague–Dawley rats (body weight, 300 to 320 g; Clea Japan, Tokyo, Japan) were fasted overnight to decrease the concentration of liver glycogen and then sacrificed. The liver was excised and washed in ice-cold homogenizing buffer containing 0.32 mol/L sucrose, 1 mmol/L dithiothreitol (Wako Pure Chemical Industries), and 20 mmol/L potassium phosphate, pH 6.5. The following procedures were all carried out at 4°C. The tissue was minced with a pair of scissors and then homogenized in homogenizing buffer using a Potter-Elvehjem grinder. The homogenate was then centrifuged at 10 000 g for 10 min. The resulting pellet was washed with two pellet volumes of homogenizing buffer and centrifuged again. The supernatants from the two centrifugations were combined and further centrifuged at 105 000 g for 1 h. The resulting pellet (microsomes) was suspended in homogenizing buffer and centrifuged again. The microsomes were resuspended in homogenizing buffer. The microsome suspension was divided into small aliquots and stored at −80°C. The microsomes were diluted with 40 mmol/L potassium phosphate, pH 6.5, immediately before use. Unlike human liver, rat liver microsomes have the 5α-R1 but not the 5α-R2 isoform.

Isopropyl-β-D-thiogalactopyranoside (IPTG) was purchased from BLD Pharmatech Inc. (Shanghai, China). Imidazole and dithiothreitol (DTT) were purchased from Wako Pure Chemical Industries (Osaka, Japan). Other chemicals used in this study were purchased from Sigma-Aldrich (St. Louis, MO, USA), Nacalai Tesque (Kyoto, Japan) or Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). All oligo DNA primers were purchased from Eurofins Genomics Inc. Japan (Tokyo, Japan). M. marburgensis was provided by RIKEN BRC through the National BioResource Project of MEXT/AMED, Japan.