Fig. 2.
Expression and purification of human mincle CRD. (A) Expression of the mincle CRD with a bacterial signal sequence followed by direct purification of folded protein released from the cytoplasm. Purification was attempted on 10-mL columns of immobilized sugars that were washed with 14 mL of Ca2+-containing buffer and then eluted with 1 mL fractions of EDTA-containing buffer. Aliquots of the elution fractions were analyzed on SDS–polyacrylamide gels that were stained with Coomassie blue. (B) Purification of mincle CRD with a biotin tag following solubilization in guanidine and renaturation. Purification on trehalose-Sepharose was performed as in (A). Parallel gels were stained with Coomassie blue or blotted onto nitrocellulose and probed with alkaline phosphatase-conjugate avidin.