Cloning, expression and purification of yeast Ent117–186. Yeast Ent1 was expressed from the T7 promoter of a modified pMBP-Ent1 vector as a His6-MBP fusion. (a) Essential elements of the expression vector. (b) Coomassie Blue-stained SDS–PAGE of the Ent117–186 purification steps. Lane M, molecular-mass marker (labelled in kDa). Lane 1, total extract of BL21 cells expressing Ent1. Lane 2, purification on Ni2+ affinity matrix. Lane 3, PreScission protease-cleaved protein. Lane 4, flowthrough of the SP column. Lane 5, elution from the SP column. Lane 6, elution from the size-exclusion column.