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Journal of Bacteriology, September 1999, p. 5855-5859, Vol. 181, No. 18
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Study of the Interaction between Bacteriophage T4 asiA and Escherichia coli sigma 70, Using the Yeast Two-Hybrid System: Neutralization of asiA Toxicity to E. coli Cells by Coexpression of a Truncated sigma 70 Fragment

Umender K. Sharma, Sudha Ravishankar, Radha Krishan Shandil, P. V. K. Praveen, and T. S. Balganesh*

AstraZeneca Pvt. Ltd., Bangalore-560 003, India

Received 2 April 1999/Accepted 2 July 1999

The interaction of T4 phage-encoded anti-sigma factor, asiA, and Escherichia coli sigma 70 was studied by using the yeast two-hybrid system. Truncation of sigma 70 to identify the minimum region involved in the interaction showed that the fragment containing amino acid residues proximal to the C terminus (residues 547 to 603) was sufficient for complexing to asiA. Studies also indicated that some of the truncated C-terminal fragments (residues 493 to 613) had higher affinity for asiA as judged by the increased beta -galactosidase activity. It is proposed that the observed higher affinity may be due to the unmasking of the binding region of asiA on the sigma protein. Advantage was taken of the increased affinity of truncated sigma 70 fragments to asiA in designing a coexpression system wherein the toxicity of asiA expression in E. coli could be neutralized and the complex of truncated sigma 70 and asiA could be expressed in large quantities and purified.

* Corresponding author. Mailing address: AstraZeneca Pvt. Ltd., 277, T. Chowdaiah Road, Malleswaram, Bangalore-560 003, India. Phone: 91 80 3340372. Fax: 91 80 3340449. E-mail: Tanjore.Balganesh{at}astra.in.astra.com.

Journal of Bacteriology, September 1999, p. 5855-5859, Vol. 181, No. 18
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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