Cm sepharose column

Expression of the recombinant FCR3-DBL3X-DBL4ε double domain has been previously described22 (link). The synthetic gene, from which DBL3X-DBL4ε DNA was amplified, carried mutations at potential N-glycosylation sites to allow production of the recombinant protein in eukaryotic, as well as bacterial, expression systems. N-glycosylation mutations were as follows: N1222Q, N1290Q, S1430G, S1594L, T1746A, T1751P, T1846A, N1916Q. The protein was expressed in E. coli SHuffle^® strain of (Novagen). Cells were centrifuged and resuspended in lysis buffer (20mM Tris-HCl, 150 mM NaCl, pH8) and lysis was performed with an Emulsiflex homogeniser (Avestin). Lysate was cleared by centrifugation at 8000 g during 1 h. FCR3-DBL3X-DBL4ε was first captured on a heparin column and eluted with 20mM Tris-HCl, 1M NaCl, pH8. The eluate was loaded on a metal affinity column (TALON, Clontech) and FCR3-DBL3X-DBL4ε recovered with 20 mM Tris-HCl, 150 mM NaCl, 200 mM Imidazole, pH8. A final purification step to remove possible impurities and aggregates was carried out by gel filtration (S75 16/60, GE Healthcare). In order to improve crystal quality, the protein was subsequently subjected to an additional purification step on a CM-Sepharose column (GE Healthcare) equilibrated in 20 mM HEPES pH 7, 40 mM NaCl. The protein was eluted by increasing NaCl concentration by 5% (w/v) steps and concentrated to 9 mg/ml.

Around 250 mg of B. mattogrossensis venom was eluted in 1.2 mL of sterile deionized water and centrifuged at 1,530 ×g for 10 minutes and then submitted to cation exchange chromatography in an Akta purifier 10 (GE-Healthcare) using a CM-Sepharose column (27 × 300 mm, GE Healthcare), equilibrated with 50 mM ammonium bicarbonate buffer, pH 8.0, and eluted with a linear gradient of 0–100% 500 mM ammonium bicarbonate, pH 8.0 at a flow rate of 5.0 mL/minute. The fractions were monitored at an absorbance of 280 nm, collected manually, identified, lyophilized, and stored at −20°C. All fractions were submitted to SDS-PAGE and enzymatic activity analyses. The isolation of the three PLA₂s was obtained by liquid chromatography using a Discovery C18 column (25 × 4.6 mm, Supelco) equilibrated with deionized water with 0.1% trifluoroacetic acid (v/v) and eluted with ten volumes of a linear gradient of 0–100% acetonitrile with 0.1% trifluoroacetic acid (v/v) at a flow rate of 1.0 mL/minute. The elution was monitored at an absorbance of 280 nm, manually collected, lyophilized, and stored at −20°C. In order to determine protein concentration, the Bradford method (Bio-Rad) was used with bovine serum albumin (BSA) as a standard [14 (link)].

BthTX-I was purified from B. jararacussu venom by the Laboratory
of Toxinology of the School of Pharmaceutical Sciences of Ribeirão Preto
(USP).
B. jararacussu crude venom (150 mg) was fractionated by
size-exclusion chromatography in a Shephacryl S-100 as described by Carone
et al. [14 (link)]. The
eluted fractions was monitored for absorbance at 280 nm, pooled, desalted in a
Hi-prep 26/10 desalting column, and lyophilized. The fraction, denominated
SPIII, was identified via SDS PAGE by containing phospholipases and myotoxins,
which have molecular masses of approximately 14 to 17 kDa. For further
purification, SPIII was submitted to ion exchange chromatography in a CM
Sepharose column (40 x 2 cm; Amersham, GE Healthcare Life Science, USA),
previously equilibrated with 50 mM ammonium bicarbonate pH 8 (Buffer A). Elution
started with the same buffer, followed by a linear gradient of 500 mM ammonium
bicarbonate pH 8 (Buffer B). The procedure was performed at a flow rate of 1.5
mL/min, with fractions of 4 mL collected based on absorbance at 280 nm. The
fraction corresponding to BthTX-I was collected, lyophilized and stored at 4°C
for subsequent analysis. The purity of BthTX-I was also assayed by 12% SDS-PAGE
and by N-terminal amino-acid sequence, using automated Edman degradation in an
automatic protein sequencer (PPSQ 33A, system, Shimadzu).