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Properties of Recombinant Bacteriophage T4 Tail Sheath Protein and Its Deletion Fragments

T. A. Kuznetsova1, A. V. Efimov1, L. G. Aijrich1, I. Yu. Kireeva1, E. I. Marusich1, P. Cappuccinelli2, P. Fiori2, P. Rappelli2, L. P. Kurochkina1,3*, B. F. Poglazov1, and V. V. Mesyanzhinov1,3

1Bach Institute of Biochemistry, Russian Academy of Sciences, Leninskii pr. 33, Moscow, 117071 Russia

2Institute of Microbiology and Virology, Sassari University, 07100 Sassari, Italy, Vialc S. Pietro 43/B

3Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow, 117871 Russia; fax: (095) 336-6022; E-mail: vvm@ibch.siobc.ras.ru

* To whom correspondence should be addressed.

Received October 29, 1997
A vector for expression of recombinant bacteriophage T4 tail sheath protein (gp18) under control of phage T7 promoter in Escherichia coli cells has been constructed. The entire length recombinant gp18 (659 amino acids) polymerizes in vivo into extended polysheaths. To study gp18 folding mechanisms, six vectors for expression of deletion mutants have been constructed. Three proteins--1N, 2N, and 3N--contain, respectively, 268, 316, and 372 amino acids of the gp18 N-tail region. The other three fragments--1C, 2C, and 3C--contain, respectively, 455, 356, and 288 amino acids of the gp18 C-tail. The fragments 1N, 2N, 1C, 2C, and 3C form insoluble aggregates during expression. However, fragment 3N accumulates in soluble form in the cellular cytoplasm and does not form polymeric structures; this has allowed an effective purification method to be developed for it. The interaction of monoclonal antibodies against recombinant gp18 with protein fragments and with phage sheath before and after contraction has been studied. The fragment 3N seems to be a stable domain of native phage sheath gp18.
KEY WORDS: bacteriophage T4, contractile tail, gp18, folding, protein engineering, expression vector, mutagenesis