Biotechnology Techniques Vol 4 NO 6 379-334 (1990) Received 17th August COMPARISION OF THE METHODS AVAILABLE FOR THE PURIFICATION OF HYBRID /3-GALACTOSIDASE PROTEINSPRODUCED UNDER THE CONTROL OF AN ANAEROBICALLY-INDUCED PROMOTER S.B. Mohanr, N. Woods2, A. Lyddiatt', & J.A. Cole 2* 1 School of Chemical Engineering 2 School of Biochemisry University of Birmingham, P 0 Box 363, Birmingham B15 2TT, UK. Summary Various methods available for purifying B-galactosidase fusion proteins were compared in an attempt to purify a hydrophobic hybrid protein, NirC"LacZ, produced under the control of an anaerobically-induced promoter. Conventional ion-exchange techniques and affinity chromatography on p-aminobenzyl-thiogalactoside Sepharose CL-4B, supplied by Sigma Chemical Co., were unsatisfactory. In contrast, anti-B-galactosidase ProtosorbTn, immunoaffinity chromatography on obtained from Promega Biotech, or with immunoadsorbents prepared in our laboratory, produced a purified hybrid protein which was suitable for N-terminal amino acid analysis. Introduction Escherichia coli B-galactosidase, which is a stable enzyme and easy to assay, is widely used for gene fusions to study the expression and properties of proteins which are difficult to assay or identify (Steers and Cuatrecasas, 1974). Various cloning vectors are available which allow genes of interest to be fused to the 1acZ gene; the resulting fusion proteins usually retain their enzyme activities thereby facilitating studies of the fused proteins (Casadaban and Cohen, 1980). Native B-galactosidase from a number of sources and hybrid proteins from E. cofi have been purified using a variety of standard techniques (Fowler and Zabin, 1983; Bulow, 1987; Mutoh et al., 1988, Nielson et al., 1988; Priyolkar et al., 1989). The affinity ligand p-aminophenyl-thiogalactoside coupled to Sepharose CL-4B has also been used successfully as a single-step purification of fusion proteins (Steers et al,. 1971; Ullman, 1984). Other authors however, did not find this technique suitable and used gel filtration on Sephacryl S-200 instead (Nielson et al., 1988). Finally, anti-/3-galactosidase antibodies have been used as affinity ligands (Schoel and Kaufmann, 1988) though the purification involved a second ion-exchange step. The nirB operon of Escherichia coli K-12 consists of five genes controlled by the transcription activator protein FNR (Peakman et al., 1990a). The first gene in the operon is nirB. Its product, the NADH-dependent nitrite reductase (EC1.6.6.4). has been thoroughly characterized (Jackson et al., 1981). Large quantities of this protein are produced under the control of the 379