Purification of Recombinant Galectins Expressed in Bacteria.

STAR protocols (2020-12-31)
Cecilia Arahí Prato, Julieta Carabelli, Valentina Cattaneo, Oscar Campetella, María Virginia Tribulatti
ABSTRACT

Galectins are soluble lectins that participate in many physiological and pathological functions. Since they can act extracellularly, the use of the recombinant protein is a recurrent strategy for studying their biological functions. Here, we provide a general protocol for the production of Galectins and their isolated or chimeric domains. We take advantage of their lectin activity and the 6xHis-tag addition for purification, thus obtaining a highly pure and active Galectin to use in both in vitro and in vivo assays. For complete details on the use and execution of this protocol, please refer to Cattaneo et al. (2011), Tribulatti et al. (2012), and Prato et al. (2020).

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Sodium chloride, BioXtra, ≥99.5% (AT)
Sigma-Aldrich
Lysozyme from chicken egg white, lyophilized powder, protein ≥90 %, ≥40,000 units/mg protein
Sigma-Aldrich
Ammonium persulfate, for molecular biology, suitable for electrophoresis, ≥98%
Sigma-Aldrich
Sodium dodecyl sulfate, ACS reagent, ≥99.0%
Sigma-Aldrich
IPTG, ≥99% (TLC), ≤0.1% Dioxane
Sigma-Aldrich
Sodium hydroxide, BioXtra, ≥98% (acidimetric), pellets (anhydrous)
Sigma-Aldrich
Phenylmethanesulfonyl fluoride, ≥98.5% (GC)
Sigma-Aldrich
Deoxyribonuclease I from bovine pancreas, Type IV, lyophilized powder, ≥2,000 Kunitz units/mg protein
Sigma-Aldrich
Triton X-100 solution, BioUltra, for molecular biology, ~10% in H2O
Sigma-Aldrich
Yeast Extract, for use in microbial growth medium
Sigma-Aldrich
Iodoacetamide, ≥99% (NMR), crystalline
Sigma-Aldrich
Sodium azide, BioUltra, ≥99.5% (T)
Sigma-Aldrich
Bovine Serum Albumin, lyophilized powder, crystallized, ≥98.0% (GE)
Sigma-Aldrich
Trizma® hydrochloride, BioUltra, for molecular biology, ≥99.0% (AT)
Sigma-Aldrich
D-Lactose monohydrate, BioUltra, ≥99.5% (HPLC)
Sigma-Aldrich
α-Lactose-Agarose, saline suspension
Sigma-Aldrich
EZMix Tryptone, Pancreatic digest of casein