Purification of recombinant human galectin-3(Gal-3)

 Inoculate 3 litters of LB-Ampicillin with BL21(DE3)-hGal-3 ^3)–6).

 Incubate overnight with agitation 225 rpm at 37°C (OD at 600nm reach to 0.7 to 1.2).

 Add IPTG at final concentration 1 mM and incubate 3 h at 37°C.

 Spin at 5,500 rpm for 20 min.

--Following steps should be done in ice or at 4°C—

 Discard the supernatant (remove the supernatant as much as possible by good draining).

 Resuspend the pellet (10 mL for each 1 L bacteria culture) with ice cold buffer (22 mM Tris-HCl (pH 7.5)-5 mM EDTA-1 mM DTT).

 Immediately add protease inhibitor cocktail 150 μL / 30 mL suspension.

 Begin prewash lactosyl column (5 mL) with 100 mL of column buffer (50 mM Tris-HCl pH 7.2, 105 mM NaCl).

 (Important—on ice) Sonicate the cell suspension at 120W for 30 sec (1 min interval between sonication) on ice for 8 times.

 Spin at 35,000 rpm for 30 min at 4°C using the rotor T70.1 in ultracentrifuge.

 Apply the supernatant to the prewashed α-Lactose agarose column.

 Wash column with 10 volumes (50 mL) of column buffer.

 Elute Gal-3 with 7 mL 150 mM lactose-column elution buffer.

• First close the cap and add 3 mL lactose buffer and rotate for 15 min, then collect the elution buffer in 1mL fraction.
• Then apply remaining 4 mL elution buffer to the column and continue to collect fractions.
• Identify the fractions containing Gal-3 by using Bradford protein assay, then pool positive fractions.

 Pass Gal-3 on Hiprep desalting PD10 column using PBS(-) 1X as a buffer.

• Collect 1mL fractions Gal-3-positive fractions are between fractions 8–20.
• Identify the fraction containing Gal-3 by using Bradford protein assay then pool positive fractions.
• Be careful: if you extensively pool Gal-3 fractions, especially try to include fractions close to the fractions of salt elution, you might as well find some lactose in the pooled ones which reduces its lectin activity.

 Concentrate on Amicon Centricon Plus-20 (10,000 MWCO), if necessary.

 Pass Gal-3 through acticlean EtOX column as many times as necessary to achieve endotoxin level less than 10 EU/mg (see step 21 for more explanation about endotoxin contamination).

 Filter sterile Gal-3 with Millex GV (Millipore Cat. #SLGV033RS).

 Determine the protein concentration with Bradford.

 (Quality control –SDS-PAGE) The purity of Gal-3 can be estimated by using a 12% SDS-polyacrylamide gel electrophoresis with ~10 μg of protein. The molecular weight of Gal-3 is approximately 30 kDa.

 (Quality control-hemagglytination assay) To estimate the activity of purified Gal-3 as a lectin, hemagglutination assay has to be routinely perform before its use (see methods in this website as well).

 (Quality control-endotoxin contamination) Endotoxin contamination should also be routinely checked by LAL assay. It should be less than 10 EU/mg with LAL kit from Lonza. We have tried both Lonza and Associated of Cap Code LAL kits (in case of false positives caused by a possible β-glucan contamination) and we obtained the same results for contamination level.

 Gal-3 should be kept in principal at 4°C. Gal-3 tends to become cleaved with time and loses its activity. Prior to use, check the activity with hemagglutination assay and cleavage level with a SDS-PAGE when use after 2 weeks kept at 4°C.

 Please note that unlike galectin-1, Gal-3 is stable in the absence of any reducing agents. Since some commercially available galectins contain significantly high concentration of reducing agents, such as DTT or mercaptoethanol, special caution should be paid when such reagents are used for biological assays.

 (Control-lectin activity) It is important to verify if the observed biological effect by Gal-3 is really due to its lectin activity. The lectin activity of galectins can be readily inhibited by β-galactoside-containing sugars, such as lactose (Galactoseβ1-4glucose) or other appropriate β-galactoside-containing oligosaccharides.

• Lactose is one of the most used inhibitors for galectins. In our laboratory, 50–150 mM lactose is routinely used to inhibit its lectin activity.
• When high concentrations of saccharide are used for cell assays, reduce salt (NaCl) concentration to achieve appropriate isotonicity (i.e., 317 mOsm/L) of the medium since cells exposed to hypertonic solutions would become fragile to any successive treatment.