Akta fplc

SEC was performed on a Superdex 200 HR 10/30 column equilibrated with 50 mM HEPES (pH 8.0) buffer containing 100 mM NaCl, at a flow rate of 0.5 mL/min (AKTAFPLC; Amersham Biosciences, Piscataway, NJ, USA), as described previously [10 (link)]. Protein (Abs_280nm) peaks were pooled and concentrated using Centricon YM-30 (Millipore Corp., Bedford, MA, USA).

GST (Glutathione S-transferase)-tagged recombinant proteins (GST-APC fragments and GST-SNAP-APC fragments) were expressed in E. coli BL21 strain at 16 °C for 20 h in the presence of 0.1 mM IPTG (Sigma), purified with glutathione sepharose (GE Healthcare) in protein lysis buffer (50 mM Tris–HCl pH 7.5, 300 mM NaCl, 0.1% Nonidet P-40, 100 µg/mL RNase, 0.5 mM PMSF). GST tag was removed by PreScission enzyme in cleavage buffer (50 mM Tris–HCl pH 7.5, 300 mM NaCl, 1 mM DTT). The proteins were further purified by Size-exclusion chromatography on an AKTA-FPLC (Amersham BioSciences) equipped with a Superose 6 Increase 10/300 GL column (GE Healthcare). Fractions were checked with SDS-PAGE and coommassie blue staining. Fractions containing the protein were collected, concentrated to a final concentration of 100 µM, snap-froze in liquid nitrogen and store at -80 °C for future use. For SNAP-tagged proteins, the SNAP tag was labeled before storing with SNAP-Surface 549 nm (New England Biolabs) according to manufacturer’s manual.

Extracted crystallin proteins were separated using size exclusion chromatography (SEC). Separation was performed using an AKTA FPLC (Amersham Biosciences, Little Chalfont, UK). Aliquots of homogenate (1 mL) were run over a Sephacryl 300 Highprep 26/60 column (Amersham Biosciences). The composition of semi-purified crystallin stocks was assessed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with a wide range marker (Sigma) and only non-contaminated samples used for further assays.

To express rHO‐1 efficiently in bacteria, we used the ΔrHO‐1 DNA sequence with prokaryotic codons and deletion of membrane anchor region coding the 22 C‐terminal residues. The sequence synthesized by Biomed (Beijing, China) was inserted into the NdeI and HindIII sites of the pMW172a plasmid, which contains the phage T7 promoter. The pMW172/ΔrHO‐1 plasmid was transformed into Escherichia coli BL21 (DE3) cells to express the ΔrHO‐1 protein, whereas the empty vector pMW172 was used as a control. To determine the optional expression condition, we compared the expression of ΔrHO‐1 at 37 °C and 200 rpm for 16 h, at 37 °C and 200 rpm and 0.1 mmol·L⁻¹ isopropyl thio‐β‐d‐galactoside for 16 h, and at 37 °C and 120 rpm for 16 h. Then, the ultrasonicated bacteria expressing ΔrHO‐1 were centrifuged at 18 000 g for 15 min at 4 °C. The ΔrHO‐1 protein was precipitated, respectively, from the supernatant at concentrations of 5–15%, 15–25%, 25–35%, 35–45%, 45–55% and 55–65% of (NH4)₂SO4 at 4 °C for 3 h before the dissolved precipitation with PBS was graded using Sephacryl S‐200HR sieve chromatography (GE Healthcare, Milwaukee, WI, USA) at A₂₈₀ by means of AKTA FPLC (Amersham Pharmacia, Piscataway, NJ, USA). The effects of purification for ΔrHO‐1 protein were analyzed by 12% SDS/PAGE.

Prediction of the quaternary structures was performed by fast protein liquid chromatography (AKTA FPLC, Amersham Biosciences) using a Superdex G200 column run at 0.5 ml/min with running buffer (50 mM NaCl, 20 mM Na₂PO₄ (pH 7.0)). The proteins were resuspended in running buffer and loaded onto the column according to the manufactures instructions. Proteins used as molecular weight markers were β-amylase (200 kDa), alcohol dehydrogenase (150 kDa), albumin (66 kDa) and carbonic anhydrase (29 kDa) which were also resuspended in the same running buffer. These were used to plot the log of their MW against retention time to generate a standard curve. Protein molecular weights were approximated based on the retention time using the standard curve generated.

Chromatographic separation of the two PGI isoforms was conducted using an AKTA FPLC from Amersham Pharmacia Biotech. Protein extracts of WT and pgi1–3 leaves (see above) were loaded onto a HiLoad 16/10 Q-sepharose HP anion exchange column (Amersham Pharmacia Biotech) equilibrated with 50 mM HEPES (pH 7.5). After washing the column, the adsorbed proteins were eluted with a linear 0–0.8 M NaCl gradient in 50 mM HEPES (pH 7.5). The flow rate was 5 ml/min and 2.5 ml fractions were collected. Fractions were analyzed for PGI activity as described above.