Purification of PAB1135. (A) Scheme of the protein expression and purification method used. (B) Purification of His-PAB1135 by affinity chromatography in Ni-NTA column. (C) Cleavage of His-PAB1135 with thrombin for releasing the His-tag. (D) Limited proteolysis of His-PAB1135 with trypsin. The His-tag and a few amino acids are released from the N-terminus of PAB1135, originating a very stable protein. Coomassie-stained 15% polyacrylamide gels. M, molecular mass marker; N, extract from non-induced cells; I, induction of His-PAB1135 expression after addition of IPTG; FT, flow through; W, wash; elution with imidazole. (E) Immuno blot with anti-His antibody detects only His-PAB1135. (F) PVDF membrane stained with coomassie brilliant blue. M, molecular mass marker. PAB1135 was obtained after incubation of His-PAB1135 with trypsin for two hours.