1068-1620/01/2704- $25.00 © 2001 MAIK “Nauka /Interperiodica” 0225 Russian Journal of Bioorganic Chemistry, Vol. 27, No. 4, 2001, pp. 225–231. Translated from Bioorganicheskaya Khimiya, Vol. 27, No. 4, 2001, pp. 257–264. Original Russian Text Copyright © 2001 by Vorobiev, Ponomarenko, Durova, Kozyr, Demin, Kolesnikov, Sashchenko, Karpeisky, Gabibov. 1 † INTRODUCTION Onconase, a ribonuclease from the Rana pipiens frog oocytes (PID g464649), is a cytotoxic protein undergoing clinical trials as an antitumor agent. This protein, earlier referred to as P-30, belongs to the super- family of RNase A [1, 2] and, along with other amphib- ian ribonucleases, can be isolated into a separate group [3]. Studies of onconase revealed a number of struc- ture–function features of this enzyme determining its cytotoxic properties: (1) Lack of interaction with inhibitors of pancreatic ribonucleases [2]. (2) Unusual resistance to proteolysis both in vitro [4] and upon incubation in transformed cell culture [3]. (3) An additional synapomorphic disulfide bond, which is not encountered in onconase-homologous ribonucleases; this bond blocks the C-terminus of the protein [5] and, possibly, the spontaneous posttransla- tional transformation of the N-terminal Gln to the pyro- glutamate (<E) [6]. (4) Some affinity of onconase toward the cytoplas- mic membrane of mammalian transformed cells [2] with the simultaneous lack of lectin properties [3]. Primary structure of onconase (Onc) was deter- mined by protein chemistry methods on the basis of complete sequencing of its peptide fragments [1] and further confirmed by cloning and sequencing of the onc 1 To whom correspondence should be addressed; fax: +7 (095) 310-7007; e-mail: ivanv@ibch.ru.) † Deceased. gene [7]. The biological function of onconase and other analogous amphibian ribonucleases is not known. It is believed that these proteins are involved into the host defense system and are toxic for mammalian predators [3]. As the exhaustive studies of the onconase properties required mutant forms of the protein, E. coli expression systems for the onc gene were developed [6, 8, 9] ensuring preparation of the recombinant onconase in an insoluble form as inclusion bodies followed by protein refolding. When the recombinant onconase so isolated was identical in primary structure to the native protein [9], their both catalytical and cytotoxic properties also coincided. At the same time, recombinant form of protein [M(-1)M23L]Onc, containing an additional Met resi- due in position –1 and a conservative substitution of Leu for Met23, possessed substantially reduced enzy- matic and cytotoxic activities [6]. The following replacement of Glu1 by Ser or Tyr partially restored the enzyme activity [10]. The chemical elimination of the additional Met residue (–1) with the remaining M23L substitution led to a protein form with a double enzyme activity and a somewhat decreased cytotoxicity. These data imply that the normal functioning of the enzyme requires the presence of a pyroglutamyl residue in position 1. However, no perceptible change in the enzyme properties was observed for recombinant forms of other pancreatic type ribonucleases with an additional N-ter- minal Met residue, in particular, for angiogenin [11]. Thus, inactivation of recombinant onconase upon intro- Structure–Function Study of Recombinant Onconase Variants I. I. Vorobiev*, 1 N. A. Ponomarenko*, O. M. Durova**, A. V. Kozyr*, A. V. Demin*, A. V. Kolesnikov*, L. P. Sashchenko***, M. Ya. Karpeisky † ****, and A. G. Gabibov* *Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul Miklukho-Maklaya 16/10, GSP Moscow, 117997 Russia **Vladimirsky Moscow Regional Research Clinical Institute, Moscow, Russia ***Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia ****Engelhardt Institute of Molecular Biology, Moscow, Russia Received November 3, 2000; in final form, January 30, 2001 Abstract—A method for expression of an onconase gene leading to a soluble form of the protein was devel- oped. The enzymatic and cytotoxic properties of the protein’s recombinant forms were studied. Recombinant onconase with an additional N-terminal Met residue isolated in non-denaturing conditions did not substantially differ from the native enzyme in ribonucleolytic activity. The addition of a 33-mer peptide containing auxiliary elements for the simplification of isolation and detection of the recombinant protein did not affect the enzyme properties of onconase. The method proposed is useful for the onconase structure–function relation studies and enables construction of onconase-based fusion proteins for anticancer therapy. Key words: ribonucleases, cytotoxicity, protein expression