Production of recombinant murine eotaxin
(A) Eotaxin was engineered for expression in bacteria by placing an amino-terminal tag containing six histidines (box with His6) and a factor Xa consensus sequence (box with Xa) from the mature sequence. The carboxy-terminal sequence contained two additional amino acids (black box). Factor Xa digestion resulted in cleavage of the tagged-construct at two sites (designated A and B with the arrows). (B) Comassie stained 12.5% SDS-PAGE (Tris/Tricine) of eotaxin protein. Low-molecular weight standards (Lane 1) and their values in kD are indicated to the left. Eotaxin was purified by Nickel and reverse phase HPLC chromatography (Lane 2) and subsequently subjected to Xa digestion. Subsequent HPLC analysis resulted in the separation of two different cleavage products designated A (Lane 5) and B (lane 3) and a mixture of the two (Lane 4). (C) N-terminal amino acid sequence analysis for the samples in Lanes 3 and 5 is indicated.