Treos 2

For characterization by SEC-MALS, purified mCes2c (1.5 mg/mL) was centrifuged at 12,851× g for 15 min at 4 °C prior the actual experiment. mCes2c was eluted from a Superdex 200 Increase 10/300 GL column (GE Healthcare) in a buffer B containing 20 mM potassium phosphate, pH 7.4 and 150 mM NaCl with a flow rate of 0.3 mL/min at 8 °C directly coupled to a MALS unit from Wyatt Technologies (TREOS II) equipped with three detection angles. Data were analyzed with the program ASTRA 7.1.2.5 (Wyatt Technologies). To determine the molecular weight based on the SEC, protein standard from Bio-Rad (Gel filtration Standard, 1511901) was used (equation for linear regression: y = −0.1963x + 4.6251; R² = 0.9984).

The SEC-MALS experiments were performed similar to as described previously^{31 (link)}. 2 mg/ml of M_i alone and in complex with 1:1 molar ratio of heparin (AMSbio, AMS.LMW Heparin) were filtered through a 0.22 μm PES filter before 100 μL each was applied to a Superdex 200 Increase 10/300 column (GE) equilibrated in 1xPBS (sigma 45ZP17) with 1 mM TCEP. The column was in line with a Shimadzu UV detector, a Wyatt TREOS II light-scattering detector, and a Wyatt Optilab tREX differential-refractive-index detector. The flow rate was 0.5 ml/min. The data were analyzed with Wyatt’s ASTRA software version 7.1.0.29. SEDFIT was used to calculate the dn/dc of the protein.

The oligomeric states of the DBL-homolog enzymes were analyzed by SEC-MALS (Treos II, WYATT Technology France) (49 (link)). The experiments were performed using a HPLC Bio-inert Shimadzu Prominence LC-20Ai (SHIMADZU Benelux B.V) coupled to a SPD-20A UV/VIS detector and a RID-20 refractive index detector. The different active fractions isolated by size exclusion chromatography were dialyzed against a “SECMALS-PBS buffer” (Na₂HPO₄ 10 mM, KH₂PO₄ 1.8 mM, NaCl 137 mM, KCl 2.7 mM pH 7.4). Samples (100 μL OXAVL02 or OXAVL06 at 0.5 to 1 mg/mL) were loaded onto a Superdex 200 Increase 10/300 G column (GE Healthcare Bio-Sciences AB Uppsala) pre-equilibrated with the “SECMALS-PBS buffer.” The column was calibrated by using bovine serum albumin (BSA) (MM = 66,430 Da) as reference standard. The data acquisition of molecular mass, distribution of Monomer-Dimer equilibrium, and percentage of aggregates were estimated using the ASTRA software (49 (link)).