Expression of recombinant human L-glutaminase in Escherichia coli: polyclonal antibodies production and immunological analysis of mouse tissues Jose ´ A. Campos, J. Carlos Aledo, Juan A. Segura, Francisco J. Alonso, Pedro M. Go ´mez-Fabre, Ignacio Nu ´n ˜ez de Castro, Javier Ma ´rquez * Laboratorio de Quı ´mica de Proteı ´nas, Departamento de Biologı ´a Molecular y Bioquı ´mica, Facultad de Ciencias, Universidad de Ma ´laga, Ma ´laga 29071, Spain Received 26 July 2002; received in revised form 6 January 2003; accepted 14 January 2003 Abstract The first complete sequence of human L-glutaminase was deduced from breast cancer glutaminase cDNA cloned in our laboratory. This cDNA clone has now been engineered to synthesize both precursor and mature forms of the protein in Escherichia coli. Among several different plasmid constructions, the expression system based on phage T7 promoter (vector pET-3c) was found to be the most efficient for glutaminase overproduction. Upon induction, precursor glutaminase accounts for about 25% of total E. coli protein, whereas a lower amount (12%) was achieved for the putative mature protein. The optimal length of the translational spacer on the ribosome binding site was shown to be eight nucleotides. However, using this length of spacer, we were unable to obtain expression in the pQE vector, tagged with a 6 Â His sequence at the NH 2 -terminus, stressing the importance of the 5V -coding sequence in the expression efficiency. Although the precursor and mature recombinant forms of glutaminase were devoid of catalytic activity, the purified protein allowed us to obtain highly specific polyclonal antibodies, as shown by immunoblot analysis of mouse tissues. Furthermore, the antibodies were able to immunoprecipitate the in vitro translated enzyme using a reticulocyte lysate system; these antibodies might be a valuable tool for studies on L-glutaminase expression in mammalian tissues. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Recombinant protein expression; Human cDNA; Mitochondrion; Tumor; Breast cancer cell 1. Introduction Phosphate-activated glutaminase (PAG; E.C. 3.5.1.2) catalyses the hydrolytic deamidation of glutamine to gluta- mate and ammonia. In mammals, the enzyme needs inor- ganic phosphate for in vitro activity and plays a key role in several physiological processes, such as renal ammonia- genesis, hepatic ureagenesis and synthesis of neurotransmit- ter glutamate in brain [1]. The catabolism of glutamine has been also linked to neoplastic transformation [2]. The high rate of glutaminolysis observed in a wide variety of tumors would be essential to maintain their proliferative capacity [3]. Thus, PAG overexpression seems to be a hallmark of experimental and human tumors [4,5]. Moreover, the inhib- ition of PAG expression by antisense technology decreases tumor cell proliferation and promotes reversion of the transformed phenotype [6]. Tumor cells transfected with antisense PAG cDNA constructs were unable to grow in the peritoneal cavity of mice. In addition, these animals were able to reject further inoculations of the wild-type tumor cells, in parallel with an activation of their immune system [7]. Two different genes coding for PAG in human have been identified: one gene is located in chromosome 2 and encodes the kidney-type (K) isozyme [8,9]; the second locus is located on chromosome 12 and codes for the liver-type (L) isozyme [9]. We have previously reported the molecular cloning and sequencing of a human L-PAG 1570-9639/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S1570-9639(03)00026-8 Abbreviations: GST, glutathione-S-transferase; PAG, phosphate-acti- vated glutaminase; hPAG, human glutaminase; phPAG, precursor human glutaminase protein; mhPAG, mature human glutaminase protein; RBS, ribosome-binding site; SD, Shine – Dalgarno sequence; UTR, untranslated region; IP, immunoprecipitation; IPTG, isopropyl thio-h-D-galactoside * Corresponding author. Tel.: +34-952-132024; fax: +34-952-132000. E-mail address: marquez@uma.es (J. Ma ´rquez). www.bba-direct.com Biochimica et Biophysica Acta 1648 (2003) 17 – 23