Akta prime plus

Wild-type and mutant α subunits (780 μg) were loaded on to a HiPrep Sephacryl S-300 HR column (GE Healthcare) coupled to an AKTA Prime Plus chromatography system (GE Healthcare). Elution profiles were analyzed using Prime View evaluation software. The column was equilibrated with Buffer D (25 mM Tris-HCl, pH 7, 150 mM NaCl), the flow rate was 0.8 ml min⁻¹, and 3 ml fractions were collected. Calibration of the column was carried out using 360 μg of each of six molecular weight standards (Serva). Aliquots (15 μl) of sizing column fractions were mixed with 5× SDS sample buffer and analyzed by 12% SDS-PAGE followed by staining with GelCode blue. In addition, aliquots (50 μl) of sizing column fractions were mixed with 5× nondenaturing sample buffer and analyzed by nondenaturing 4–15% gradient precast gels followed by staining with Imperial Stain (ThermoScientific) or Pierce Silver Stain Kit (ThermoScientific). In experiments requiring the pooling of sizing column fractions, the indicated fractions were combined and concentrated down to a volume of 0.6 ml using Pierce Protein Concentrators, 9K (ThermoScientific). These pooled and concentrated fractions were then mixed with crude lysates of BL21 cells expressing untagged archaeal β subunits. Proteins were repurified by ICAR and analyzed by native PAGE and substrate-overlay assay as described above.

The coding regions for mouse HP1 were subcloned from pGEX2T, previously described in (39 (link)), into the expression vector pPROEX HTb. The vector expressing the his-tagged HP1α hinge domain 3K-A mutant, where three lysines at positions 104–106 were replaced with alanine, was created from the pPROEX HTb hisHP1α vector using standard methods.
For protein expression, the hexahistidine-HP1 expression vectors were transformed into Escherichia coli Rosetta II cells (Merck, Germany) and expression induced with 0.4 mM IPTG (isopropyl-β-D-thiogalactopyranoside). After lysis, hexahistidine-HP1 paralogs were purified using the AKTA Prime Plus (GE Healthcare, UK) chromatography system and a Ni-NTA IMAC column by elution with 500 mM imidazole, concentrated using Vivaspin 20 (5 kDa MWCO) ultrafiltration devices (GE Healthcare) before passing through a Superdex 75 10/300 GL (GE Healthcare) size exclusion chromatography column, and finally re-concentrated as performed previously.

Mature dry peanut seeds were blended into a fine powder, pigments and fats were removed by subsequent washes with acetone and hexane. The dried seed powder was extracted against buffer (50 mM Tris-HCl, pH 8.0) with 1% PVP (w/v) by gentle stirring at 4°C for overnight and the resulting supernatant is called as peanut crude proteinase inhibitor (PnCPI) extract (Prasad et al., 2010c (link)).
The clear PnCPI extract was subjected to 0-20%, 20-60%, 60-80% (NH₄)₂SO₄ fractionation. The 20-60% pellet fraction with prominent TI activity was purified by passing successively through trypsin bound CNBr Sepharose 4B column (XK 16/20, 3 cm, 60 ml/h flow rate) and Sephadex G-50 fine column (XK 16/100, 85 cm, 30 ml/h flow rate) by fast protein liquid chromatography (FPLC) system (AKTA prime plus, GE Healthcare). The eluted protein fractions (1.0 ml) from each chromatographic column were analyzed for TI activity. The fractions with significant TI activity were collected and subjected to dialysis (3 kDa c/o) followed by freeze-drying (Labconco, United States)/Amicon filters (3 kDa c/o). The purified protein with prominent TI activity was estimated for protein content and stored at −20°C for further use.

A His‐tagged ScFv (SM3E) (Graff et al., 2004) was expressed and purified from Pichia pastoris as previously described (Tolner et al., 2006) and used as the test protein for recovery from cell culture media. Ultraviolet light absorbance and conductivity were analyzed by linking the RFC to an Akta Prime Plus (GE Healthcare). Eluted fractions were resolved by 10% (v/v) sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE), stained for 1 h with Coomassie Brilliant Blue (Sigma–Aldrich) and destained until clear using a mixture of 10% (v/v) glacial acetic acid, 30% (v/v) methanol, and 60% (v/v) double distilled water (ddH₂O).

S1 protein and RBD protein recombinant plasmids were prepared using the pcDNA™3.3-TOPO^® vector (ThermoFisher, Waltham, MA. United States. Cat No. K8300-01). The vector was injected into the Chinese Hamster Ovary (CHO) cell to express the protein. After culturing the transformed CHO cells, the supernatant is collected, filtered, and purified by column loading (GE healthcare, AKTA prime plus). After purification, the protein is identified by SDS-PAGE and Western Blotting. The purified antigen is used as the vaccine antigen after checking the concentration using Nanodrop (ThermoFisher, Waltham, MA. United States. AZY2017596) (Figure 1).