Figure 2

Purification and partial proteolysis of A118 integrase. (A) SDS-PAGE of A118 integrase purification. Lanes 1 to 3, uninduced, induced, and extract after clearing spin, respectively. Lane 4, N-terminally His-tagged integrase after Ni-NTA, and then heparin-Sepharose chromatography (lane 5). Lane 6, native integrase after heparin-Sepharose chromatography, and (lane 7) His-tagged C-terminal domain (residues 158 to 452) after Ni-NTA chromatography. Lane M, molecular weight (MW) markers are 6, 14.4, 21.5, 31, 36.5, 55.4, 66.3, 97.4, 116.3, and 200 kDa. (B) Size exclusion chromatograms (Superdex 200) of full-length ^HisInt (calculated dimer MW = 110,684) and ^HisInt^158-452 (C-term, calculated monomer MW = 37,438). Size standards are chymotrypsin (25 kDa), ovalbumin (43 kDa), BSA (67 kDa), and γ-globulin 160 (kDa). (C and D) Partial proteolysis of A118 integrase by chymotrypsin and proteinase K, respectively. Integrase (3 μg) was digested with 20, 40, and 60 ng chymotrypsin or 50, 75, 100 and 125 ng proteinase K for 10 min at 23°C in the absence or presence of twofold molar excess of 50 bp attP oligonucleotides, as designated. The products were displayed on a 15% SDS-PAGE gel. Lane C, no protease control; MW markers (lane M) range from 6 to 66.3 kDa, as in (A). (E) Schematic representation of the A118 integrase domain structure based on proteolysis experiments and secondary structure prediction programs. Mutagenesis experiments have shown that the putative active site serine (S) at residue 10 within the catalytic domain is required for recombination. The long putative oligomerization helix is analogous to α-helix E in resolvase structures. bCH, a basic (11 arginines and lysines) segment between residues 274 and 314 containing four cysteines and four histidines. Chymotrypsin and proteinase K fragments identified by mass spectrometry are represented below (see text).