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Expression and Preparation of Fusion Proteins from Recombinant λgt11 Phages

  • Sheng-He Huang
  • Ambrose Jong
Protocol
  • 90 Downloads
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

The phage λgt11 system has become increasingly popular for expression of cDNAs or genomic DNAs either in phage plaques or in bacteria lysogenized with recombinant phages (1,2). It offers the advantages of high cloning efficiency, high-level expression, the relative stability of β-galactosidase fusion proteins, and simple approaches to purify the fusion proteins. After the desired clone is detected and purified, it is often necessary to obtain preparative amounts of recombinant protein specified by the fusion of the foreign sequence to the carboxyl-terminus of β-galactosidase in λgt11 expression system. The conventional method for preparing fusion proteins from the recombinant λgt11 clones involves production of phage lysogens in Escherichia coli strain Y1089 followed by inducing lacZ-directed fusion protein expression with isopropyl-β-D-thiogalactopyranoside (IPTG) (1). This method has two limitations: it is time-consuming, and phage lysogeny occurs at a low frequency. We have previously described a method for making fusion proteins from LB agar plates containing E. coli Y1090 infected with a high concentration of recombinant λgt11 phages (up to 5 × 106 PFU/150 × 15-mm plate) (3). A liquid culture method for preparing fusion proteins from E. coli Y1090 infected with the λgt11 clones has previously been described (4). More recently, some improvements have been made on the plate method by repeating induction and elution (5). Although the liquid culture method allows the recovery of only 0.2–1% of total proteins (6,7), this method generally yields 5–10% of expressed protein in solution, that is, most lysed cells are trapped in the agar and the expressed proteins are recovered in a small volume of inducing solution, resulting in a higher final concentration of protein.

Keywords

Fusion Protein Ammonium Sulfate Immunoaffinity Column Recombinant Phage Phage Plaque 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Humana Press Inc., Totowa, NJ 2000

Authors and Affiliations

  • Sheng-He Huang
    • 1
  • Ambrose Jong
    • 1
  1. 1.Division of Infectious Disease, Department of PediatricsUniversity of Southern California, Children’s Hospital of Los AngelesLos Angeles

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