Volume 212, number 2, 233-236 FEB 04442 February 1987 trp operon induction during the expression in E. zyxwvutsrqponmlkjihgfedcb coli of two IFN-y sequences E.D. Sverdlov, S.A. Tsarev, R.A. Krykbaev, I.P. Chernov and V.M. Rostapshov M.M. Shemyukin Institute of Sioorganic Chemistry, Moscow, USSR Received 9 December 1986 Two nucleotide sequences coding for mature human immune interferon (IFN-)I) and differing from each other by nine N-terminal nucleotides were expressed in E. coli under the control of a trp promoter. The longer gene variant after the ATG initiatory codon contained a TGT TAC TGC sequence, which was absent in the shorter gene. When expressed in E. cofi under the direction of identical tr~~~ption and translation regulatory elements, these genes showed different susceptibility to induction. Complementary DNA; Interferon-~; Gene variant; Gene expression; Tryptophan promoter; Tran~~ptional control 1. INTRODUCTION Expression efficiency in a host cell is a function of many factors, such as promoter efficiency, the secondary structure of the RNA to be synthesized, the Shine~D~garno sequence structure, composi- tion and the distance from the ATG codon, and of course the structure of the gene itself [l-4]. Numerous studies on foreign gene expression are now in progress, but we are still somewhat far from a true understanding of the process and the factors governing it. Here we report the expression of two gene variants coding for mature IFNy and differing in structure by nine nucleotides [5,6]. Gene expres- sion was directed by identical regulation sequences taken from the trp operon and a synthetic Shine- Dalgarno sequence. The small difference in the en- coding part of the gene was shown to affect con- siderably its expression in the cell, Correspondence address: E.D. Sverdlov, M.M. Shemyakin Institute of Bioorgauic Chemistry, Moscow, USSR 2. MATERIALS AND METHODS cDNA coding for the synthesis of IFN-y was ob- tained by reverse transcription of l~phoc~e mRNA induced by staphylococcal enterotoxin. The cDNA obtained was cloned in the P&I site of a pBR322 in E. co/i K-802 171. The sequences coding for the two variants of mature IFN-7 [5,6] were constructed using cDNA fragments and synthetic oligonucleotides contain- ing the ATG initiating codon and Shine”Daigarno sequence. Gene expression was controlled by a previously cloned trp promoter 181. Recombinant plasmids were used to transform E. coli strain MH-1. 2.2. Cultivation of bacterial cells carrying prF~-~-trp-~ and p~F~~-tr~Z E. co/i harbouring pIFN-y-trp-1 and pIFN-y- trp-2 were grown overnight in LB medium contain- ing 5 fig/ml tetracycline. The overnight culture was diluted in M-9 medium supplemented with 0.2% casamino acids and 5 pg/ml tetracycline in a 1: 100 ~bi~he~ by Eisevier Sfience Publishers 3. V. ~Bio~~~cul Division) 00145793/87/$3.50 0 1987 Federation of European Biochemical Societies 233