PL Expression System A Prokaryotic Expression System

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PL Expression SystemA Prokaryotic Expression
System
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Introduction

• Expression of heterologous proteins in E. coli.
• Foreign genes inserted into the multiple cloning
  site
• Regulated by a tryptophan-inducible expression
  system
• Utilizing the strong PL promoter from
  bacteriophage lambda
• Useful for the expression of potentially toxic
  proteins in
• E. coli.
• The PL promoter provides high-level expression
  of
• recombinant proteins.

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Regulation of Expression

• 1. In the absence of tryptophan, expression of
  the of repressor is driven by the trp promoter

2. The cl repressor protein binds to the
operator region up-stream of the PL promoter and
prevent transcription of the gene of interest

3. Trytophan is added to the medium and a
tryptophan-trp repressor complex is formed. this
complex binds tightly to the trp operator
blocking expression of the cl repressor
4. The cl repressor fails off of the PL
operatior, allowing transcription of the gene
of interest
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Features of pLEX
Feature
Benefit
High-level expression of recombinant protein
PL promoter

• Lambda cII ribosome binding
• site and initiation ATG

Efficient translation of recombinant protein
E. coli aspA transcription terminator
Efficient transcription termination of mRNA
Selection and maintenance in E. coli
Ampicillin resistance
pUC origin
Maintenance in bacteria and high copy number
Polylinker region
Cloning of the desired gene into the pLEX vector
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Positive control for expression
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Flow Chart of Experimental Process
Ligate Gene of interst into pLEX
GI724 Cells
Optimize growth of cells and expression of
reconbinant protein
Perform Restriction Analysis or Sequence AmpR
transformants
Desired clone
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Advantage

• 1. Cloning
• Polylinker region
• High copy number
• 2. Transscription
• PL pormoter / operator (conditional induction)
• E.coli aspA transcription terminator
• 3. Translation
• Lambda c? ribosome binding site and initiation
  ATG

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Disadvantage

• 1. Purification
• requirement of protein purification technique
• 2. Insoluble form
• 3. Using GI724 strain
• The specially constructed strain, GI724, for
  proper regulation of expression
• 4. The time of induction

The major variable to optimize when using the PL
expression system. increase or decrease the time
to achieve maimum levels of recombinant protein