Expression Using Vectors with Phage λ Regulatory Sequences

Allan R. Shatzman1, Mitchell S. Gross1, Martin Rosenberg1

1 SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 16.3
DOI:  10.1002/0471142727.mb1603s11
Online Posting Date:  May, 2001
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Abstract

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 first and second 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.

     
 
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Table of Contents

  • Basic Protocol 1: Temperature Induction of Gene Expression
  • Basic Protocol 2: Chemical Induction of Gene Expression
  • Support Protocol 1: Authentic Gene Cloning Using pSKF Vectors
  • Support Protocol 2: Construction and Disassembly of Fused Genes in pSKF301
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Temperature Induction of Gene Expression

  Materials
  • Expression vector (e.g., pSKF series; see support protocols)
  • E. coli AR58 or equivalent (Table 97.80.4711)
  • LB plates containing the appropriate antibiotic (unit 1.1)
  • LB medium containing appropriate antibiotic (room temperature and prewarmed to 65°C; unit 1.1)
  • SDS/sample buffer (unit 10.210.2)
  • Gyrotory air or water shaker, 32° and 42°C
  • Additional reagents and equipment for transformation (unit 1.8)

Basic Protocol 2: Chemical Induction of Gene Expression

  Materials
  • Expression vector (e.g., pSKF series; see protocol 3support protocols)
  • E. coli AR120 or equivalent (Table 97.80.4711)
  • LB plates containing appropriate antibiotic (unit 1.1)
  • LB medium containing appropriate antibiotic (unit 1.1)
  • 60 mg/ml nalidixic acid in 1 N NaOH (not necessary to filter sterilize; Table 97.80.4711)
  • Additional reagents and equipment for transformation (unit 1.8)

Support Protocol 1: Authentic Gene Cloning Using pSKF Vectors

  Materials
  • Appropriate restriction endonucleases and buffers (unit 3.1)
  • pSKF101 vector (available from A. Shatzman; Fig. )
  • Competent E. coli AS1 (Table 97.80.4711; also known as MM294cI+)
  • Additional reagents and equipment for restriction digestion, (unit 3.1), oligonucleotide synthesis and purification (units 2.11 & 2.12), nondenaturing PAGE (unit 2.7), isolation, recovery, and quantitation of DNA (unit 2.6 & 3.NaN), subcloning DNA fragments (unit 3.16), transforming, plating, and growing E. coli (units 1.8, 1.1, & 1.3), and DNA miniprep (unit 1.6)

Support Protocol 2: Construction and Disassembly of Fused Genes in pSKF301

  Materials
  • Appropriate restriction endonucleases and buffers (unit 3.1)
  • Klenow fragment of E. coli DNA polymerase I (unit 3.5)
  • pSKF301 vector (available from A. Shatzman; Figs. & )
  • T4 DNA ligase (unit 3.14)
  • Competent E. coli AS1 (Table 97.80.4711; also known as MM294cI+)
  • Additional reagents and equipment for large‐scale plasmid prep (unit 1.7), agarose gel electrophoresis (unit 2.52.5), extraction and precipitation of DNA (unit 2.12.1), transformation of competent cells (unit 1.8), and restriction digestion and mapping (units 3.1 3.3)
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Figures

Videos

Literature Cited

Literature Cited
   Casadaban, M., Martinez‐Arias, A., Shapira, S., and Chou, J. 1983. Translation initiation in prokaryotes. Methods Enzymol. 100:293‐308.
   de Boer, H.A., Comstock, L.J., Yansura, D., and Heynecker, H. 1982. In Promoters: Structure and Function (R.L. Rodriguez and M.J. Chamberlin eds.) pp. 462‐481. Praeger, New York.
   Edman, J.C., Hallewell, R.A., Valenzuela, P., Goodman, H.M., and Rutter, W.J. 1981. Synthesis of hepatitis B surface and core antigens in E. coli. Nature (London) 291:503‐506.
   Gold, L., Pribhow, D., Schneider, T., Slinedling, S., Singer, B., and Stormo, G. 1981. β‐Galactosidase gene fusions for analyzing gene expression in Escherichia coli and yeast. Annu. Rev. Microbiol. 35:365‐403.
   Guarente, L. 1983. Construction and use of gene fusions to lac Z (β galactosidase) that are expressed in yeast. Methods Enzymol. 101:167‐181.
   Rose, M. and Botstein, D. 1983. Yeast promoters and lacZ fusions designed to study expression of cloned genes in yeast. Methods Enzymol. 101:181‐192.
   Rosenberg, M., Ho, Y.S., and Shatzman, A.R. 1983. The use of pKC30 and its derivatives for controlled expression of genes. Methods Enzymol. 101:123‐138.
   Shatzman, A.R. and Rosenberg, M. 1987. Expression, identification and characterization of recombinant gene products in Escherichia coli. Methods. Enzymol. 152:661‐673.
   Studier, F.W. and Moffatt, B. 1986. Use of bacteriophage T7 RNA polymerase to direct selective high‐level expression of cloned genes. J. Mol. Biol. 189:113‐130.
   Tabor, S. and Richardson, C. 1985. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc. Natl. Acad. Sci. U.S.A. 82:1074‐1078.
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