Purification of recombinant human galectin-1(Gal-1)

 Inoculate 3 litters of LB-Ampicillin with BL21(DE3)-hGal-1 ³⁾.

 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 / 30mL suspension.

 Begin prewash lactosyl column (5 mL) with 100 mL of column buffer (50 mM Tris-HCl pH 7.2, 105mM 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-1 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 1 mL fraction.
• Then apply remaining 4 mL elution buffer to the column and continue to collect fractions.
• Identify the fractions containing Gal-1 by using Bradford protein assay, then pool positive fractions.

 Please note that among members of the galectin family, Gal-1 is a unique lectin that is sensitive to oxidation due to a cysteine residue proximal to its dimerization site ⁴⁾. Oxidation leads to its inactivation as a lectin and results in the loss of its hemagglutinin activity. We sometimes apply the method of iodoacetamidation, which makes Gal-1 resistant to atmospheric oxygen in absence of disulfide-reducing reagents and yet preserves some of its biological functions ⁵⁾. In some cases, we have begun to use this method to stabilize Gal-1, although it is highly recommended that each laboratory verifies whether this treatment has an impact on its own assays.

• Galectin-1 is resuspended in a 100 mM lactose–PBS solution to protect the CRD of the protein.
• Then, Gal-1 (2–5 mg/mL) is incubated overnight at 4°C with a final iodoacetamide concentration of 100 mM in dark.
• Free iodoacetamide and lactose are extensively removed by series of dialysis against PBS, followed by sterilization as described below. The treated protein remains stable over a prolonged period at 4°C as previously described ^{6) 7)}.
• Important note: Previously many researchers used Gal-1 with reducing agents (such as DTT or mercaptoethanol) so that its activity remains intact during assays or storage. However, many biological assays could be significantly perturbed by the presence of reducing agents ⁸⁾. Indeed, as little as 5 μM of DTT was sufficient to halt virtually all natural migration and movement of T lymphocytes (time-lapse live cell imaging, unpublished observations by CSP and SS). The presence of 1 μM of DTT also inhibited HIV-1 infection (unpublished observations by CSP and SS) ⁹⁾.

 Dialysis the Gal-1 containing fractions overnight against 4 l PBS(-). Change the buffer 4 times every 6 h.

 Pass Gal-1 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-1 protein with Millex GV (Merck Millipore Cat. # SLGV033RS).

 Determine the protein concentration with Bradford.

 (Quality control–SDS-PAGE) The purity of galectin-1 can be estimated by using a 12% SDS-polyacrylamide gel electrophoresis with ~10 μg of protein. The molecular weight of Gal-1 is approximately 15 kDa.

 (Quality control-hemagglytination assay) To estimate the activity of purified Gal-1 as a lectin, hemagglutination assay has to be routinely performed before its use ¹⁰⁾ (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-1 should be kept in principal at 4°C. Gal-1 tends to be oxidized with time and lose its activity. Thus, prior to use this lectin, check the activity by hemagglutination assay.

 (Control-lectin activity) It is important to verify if the observed biological effect by Gal-1 is 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.