UNIT 16.3 Expression Using Vectors with Phage λ Regulatory Sequences
Published Online: 1 MAY 2001
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Molecular Biology
How to Cite
Shatzman, A. R., Gross, M. S. and Rosenberg, M. 2001. Expression Using Vectors with Phage λ Regulatory Sequences. Current Protocols in Molecular Biology. 11:16.3.1–16.3.11.
- Published Online: 1 MAY 2001
- Published Print: JUL 1990
In the expression system described here, plasmids (pSKF) utilize regulatory signals-such as the powerful promoter pL -from the bacteriophage λ. Transcription from pL can be fully repressed and plasmids containing it are thus stabilized by the λ repressor, cI. The repressor is supplied by an E. coli host which contains a integrated copy of a portion of the λ genome. This so-called defective lysogen supplies the λ regulatory proteins cI and N but does not provide the lytic components that would normally lead to cell lysis. Thus, cells carrying these plasmids can be grown initially to high density without expression of the cloned gene and subsequently induced to synthesize the product upon inactivation of the repressor. This system also ensures that pL-directed transcription efficiently traverses any gene insert, which is accomplished by providing the phage λ antitermination function, N, to the cell and by including on the pL transcription unit a site necessary for N utilization (Nut site). The N protein interacts with and modifies the RNA polymerase at the Nut site so as to block transcription termination at distal sites in the transcription unit. In order to express the coding sequence, efficient ribosome-recognition and translation-initiation sites have been engineered into the pL transcription unit. Expression occurs after temperature or chemical induction inactivates the repressor (see and basic protocols). Restriction endonuclease sites for insertion of the desired gene have been introduced both upstream and downstream from an ATG initiation codon. Thus, the system allows either direct expression or indirect expression (via protein fusion) of any coding sequence, thereby potentially allowing expression of any gene insert. Protocols describe direct expression of “authentic“ gene products, as well as heterologous genes fused to highly expressed gene partners generates chimeric proteins that differ from the native form. In the latter case, the fusion partner can be removed to obtain an unfused version of the gene product.