Journal of Biotechnology 117 (2005) 263–266 Short communication Expression and characterisation in E. coli of mutant forms of saporin Eugenia Pittaluga, Anna Poma, Adele Tucci, Laura Span` o ∗ Department of Basic and Applied Biology, University of L’Aquila, Via Vetoio Coppito, 67100 L’Aquila, Italy Received 16 December 2004; received in revised form 19 January 2005; accepted 29 January 2005 Abstract In the present communication, we report on the expression and characterisation in Escherichia coli of mutant derivatives of saporin, a type 1 ribosome-inactivating protein from Saponaria officinalis L. The effects of substitution of Glu 176 with Lys and those of deletion of 19 amino acids at the C-terminal were evaluated both in vivo, testing the influence of expressed proteins on bacterial growth and in vitro measuring their N-glycosidase and supercoiled DNA relaxation activities. Results indicate that both modifications of the wild-type protein abolish its toxicity to bacterial cells and impair its enzymatic activity on polynucleotide substrates, either RNA or DNA. © 2005 Elsevier B.V. All rights reserved. Keywords: Ribosome-inactivating protein; Saporin; Saponaria officinalis; Non-toxic mutants Ribosome-inactivating proteins (RIPs) are N-glyco- sidases [EC 3.2.2.22] that remove a specific adenine from the sarcin/ricin loop of the large rRNA (Nielsen and Boston, 2001; Stirpe, 2004). RIPs have been re- ported to remove adenine also from different substrates, in particular from various forms of DNA (Barbieri et al., 1997; Girbes et al., 2004). These enzymes might have great therapeutic importance in the construction of chi- merical toxins, such as immunotoxins, useful for treat- ing various forms of cancer, autoimmune diseases and HIV infection (Bolognesi et al., 2000; Qi et al., 2004). ∗ Corresponding author. Tel.: +39 0862 433263; fax: +39 0862 433273. E-mail address: laura.spano@aquila.infn.it (L. Span ` o). Saporin is a single chain (type 1) RIP present in different tissues of the soapwort Saponaria of- ficinalis L. (Ferreras et al., 1993). We have char- acterised an active site mutant, E176K, and a C- terminal deletion mutant, K234stop, of the major seed isoform of saporin (Ceriotti, A., personal com- munication). The primer sense (5 ′ → 3 ′ GCCGCG GAGCTC GTCACATCAATCACATTA), primer an- tisense (5 ′ → 3 ′ GGGAAA CGGCCG CTGCAGT- CATGTGATTAG) and primer antisense K234stop (5 ′ → 3 ′ GGGAAA CGGCCG TCATTTTCCAAACC- CATC), containing the SacI and EagI site, were de- signed to clone saporin sequences lacking the NH 2 - extrapeptide into the expression vector pET-30 a(+) (Novagen). 0168-1656/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2005.01.021