Gene, 142 (1994) 1477151 0 1994 Elsevier Science B.V. All rights reserved. 0378-l 119/94/$07.00 147 GENE 07143 Overproduction and purification of biologically active native fungal a-sarcin in Escherichia coli zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA (Aspergillus giganteus; cytotoxins; fusion protein; ribosome-inactivating proteins; signal peptide) Javier Lacadena=, Alvaro Martinez de1 Pozoa, Jo& L. Barberob, Josh M. Manchefioa, Maria Gasset=, Mercedes Ofiaderraa, Carlos Lbpez-Otin”, Sagrario Ortegad, Jo& Garciad and Jo& G. Gavilanes” zyxwvutsrqpo “Departamento de Bioquimica y Biologia Molecular I, Universidad Complutense de Madrid, 28040 Madrid, Spain; bDepartamento de Investigacidn, Antibibticos Farma S.A., Antonio Ldpez 109, 28026 Madrid, Spain. Tel. (34-l) 589-5478; ‘Departamento de Biologia Funcionul, Universidad de Oviedo, 33006 Oviedo, Spain. Tel. (34-8) 510-4201: and *Centro de Investigaciones Bioldgicas, Consejo Superior de Investigaciones Cient$cas. Velrizquez 144, 28006 Madrid, Spain. Tel. (34-l) 564- 4570 Received by J.R. Kinghorn: 5 September 1993; Revised/Accepted: 2 October/8 October 1993; Received at publishers: 2 December 1993 SUMMARY An efficient system was developed to produce, in Escherichia coli, large amounts of native a-sarcin (c&ar), a cytotoxin from the mold A spergillus giganteus. The protein has been purified to homogeneity with a yield of 1.5 ug/ml of original culture. The constructed expression vector (pINPGc&) is based on the synthesis of a fusion protein between c&ar and a modified version of the OmpA signal peptide. This peptide seems to favour the postranslational processing of the fusion protein. The purified recombinant c.+sarcin (re-c&ar) is structurally identical to the mature fungal protein according to the following criteria: N-terminal amino acid (aa) sequence, aa composition, electrophoretic mobility, chromatographic behaviour, immunoreactivity and spectroscopic features. Indeed, the recombinant protein recovered is completely func- tional, since it cleaves, in vitro, eukaryotic rRNA and it is able to interact with phospholipid vesicles with the same specificity as the native fungal aSar. INTRODUCTION aSarcin (cLSar) is a cytotoxic protein secreted by the mold A spergillus giganteus MDH 18894 (reviewed by Wool et al., 1992). It was discovered and characterized as an antitumor agent displaying selectively its cytotoxic action (Olson and Goerner, 1965; Olson et al., 1965). This cytotoxicity is due to its ribonucleolytic activity against rRNA, inhibiting protein biosynthesis (Schindler and Correspondence to: Dr. A. Martinez de1 Pozo, Departamento de Bioquimica y Biologia Molecular I, Facultad de Ciencias Quimicas, Universidad Complutense de Madrid, 28040 Madrid, Spain. Tel. (34-l) 394-4259; Fax (34-l) 394-4159; e-mail: mayte@solea.quim.UCM.ES Abbreviations: A., Aspergillus; aa, amino acid(s); Ap, ampicillin; bp, base pair(s); cDNA, DNA complementary to RNA; DMPG, dimyristoyl- phosphatidylglycerol; DPH, 1,6-diphenyl-1,3,5_hexatriene; EF, elonga- tion factor; HPLC, high-performance liquid chromatography; IPTG, SSDI 0378-1119(94)E0739-Z Davies, 1977; Endo and Wool, 1982). This action is highly specific, since clSar cleaves only one phosphodiester bond of the 28s rRNA when native ribosomes are used as substrate (Endo et al., 1983). The cleavage site is evolu- tionary conserved. This region has an important role in ribosome function because it is involved in EF-l-dependent binding of aminoacyl-tRNA and EF-2-catalyzed GTP hydrolysis and translocation (Wool et al., 1992). However, although aSar is able to hydrolyze isopropyl-P-D-thiogalactopyranoside; kb, kilobase or 1000 bp; lacl, lac repressor-encoding gene; lpp, gene encoding lipoprotein; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; OmpA, outer membrane protein A; PAGE, polyacrylamide-gel electrophoresis; PCR, polymerase chain reaction; PolIk, Klenow (large) fragment of E. coli DNA polymer- ase I; poly(A)+RNA, polyadenylated RNA; R, resistant/resistance; r, ribosomal; re-, recombinant; c&ar, cc-sarcin; Sar, gene encoding c&ar: SDS, sodium dodecyl sulfate; ss, single strand(ed); UV, ultraviolet.