Preparation of all media and protocols for heterologous expression of aga-F75 in P. pastoris GS115 followed the Pichia expression manual (Invitrogen, Carlsbad, CA, USA). The gene fragment coding for mature Aga-F75 without the signal peptide-coding sequence was cloned into vector pPIC9 to construct the recombinant plasmid pPIC9-aga-F75, which was further linearized and transformed into P. pastoris GS115 competent cells by electroporation. Positive transformants were screened based on their α-galactosidase activities as described below. The transformant showing the highest activity was selected for large-scale fermentation in 1-l conical flasks. To purify recombinant Aga-F75, the crude enzyme was sequentially loaded onto the HiTrapTM Desalting column and HiTrap Q Sepharose XL FPLC column from GE Healthcare (Uppsala, Sweden). The purity of Aga-F75 was identified to be 90% based on SDS-PAGE. Purified Aga-F75 was deglycosylated with PNGase F following the instruction of manufacturer (New England Biolabs, Ipswich, MA, USA).
Hitrap q sepharose xl fplc column
Manufactured by GE Healthcare
Sourced in Sweden
The HiTrap Q Sepharose XL FPLC column is a chromatography column designed for fast protein liquid chromatography (FPLC) applications. The column contains Q Sepharose XL, a strong anion-exchange medium, which is used for the purification and separation of biomolecules such as proteins, peptides, and nucleic acids.
Automatically generated - may contain errors
Lab products found in correlation
Hitrap, by Cytiva (4 mentions)
Hitrap desalting column, by GE Healthcare (2 mentions)
Pichia expression manual, by Thermo Fisher Scientific (1 mentions)
Pngase f, by New England Biolabs (1 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
Pichia expression kit, by Thermo Fisher Scientific (1 mentions)
4 protocols using hitrap q sepharose xl fplc column
1
Heterologous Expression and Purification of Aga-F75 in P. pastoris
2
Purification and Characterization of Recombinant Cellulases
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The viva flow 200 ultrafiltration membrane system (Sartorius, Germany) with 5 kDa cut-off was used for the concentration and buffer exchange (to 0.1 M Tris-HCl, pH 8.0) of the crude enzymes. The recombinant proteins were desalted by HiTrapTM Desalting column and purified using the HiTrap Q Sepharose XL FPLC column (GE Healthcare) pre-equilibrated with 0.1 M Tris-HCl (pH 8.0). The gradient NaCl of 0–0.7 M at the flow rate of 3 mL/min was used to elute the proteins. Fractions showing cellulase activities were pooled and further desalted with a 5 kDa molecular cut-off concentration tube (Millipore) using 0.1 M McIlvaine buffer (pH 3.5 or 4.5). The purified proteins were separated on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for purity and molecular mass analysis. Protein concentration was determined by using the Bradford method.
3
Purification and Characterization of Xylanase
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The cell-free culture supernatants were collected by centrifugation at 12,000 g for 10 min at 4°C, and concentrated by 5 kDa cut off Vivaflow 200 ultrafiltration membrane (Vivascience, Hannova, Germany). The crude enzymes were loaded onto the HiTrap™ desalting columnand HiTrap Q Sepharose XL FPLC column (GE Healthcare, Uppsala, Sweden) equilibrated with buffer A (20 mM Tris–HCl, pH 8.0). Proteins were eluted with 1.0 M NaCl in the same buffer at a flow rate of 3 mL/min. Fractions exhibiting xylanase activity were pooled, and concentrated by 5 kDa cut off Vivaflow 50 ultrafiltration membrane (Vivascience, Hannova, Germany). SDS-PAGE was performed in a 12% (w/v) polyacrylamide gel to analyze the purified enzyme. The protein concentrations were determined by a protein assay kit (Bio-Rad, USA).
4
Recombinant NfBGL1 Expression in Pichia
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Heterologous expression of recombinant NfBGL1 was conducted following the Pichia Expression Kit (Invitrogen). The cDNA fragment of mature NfBGL1 without the signal peptide-coding sequence was digested with EcoRI and NotI, and then cloned into the corresponding sites of vector pPIC9. The recombinant plasmid pPIC9-Nfbgl1 was then linearized with BglII and transformed into P. pastoris GS115 competent cells by electroporation (Bio-Rad, Hercules, CA). The positive transformants were selected based on their enzymatic activities in shake tubes. The transformant that exhibited the highest β-glucosidase activity in culture supernatant was used for large-scale fermentation in 1-l Erlenmayer flasks. The cell-free culture supernatants were collected and loaded onto a HiTrap Q Sepharose XL FPLC column (GE Healthcare, Uppsala, Sweden) equilibrated with buffer A (20 mM McIlvaine buffer, pH 7.0). Proteins were eluted with NaCl gradients (0–1.0 M) in the same buffer at a flow rate of 3 ml/min. The fractions containing enzyme activity were collected and evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein concentration was determined by the Bradford assay with bovine serine albumin as the standard.
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