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In the following, various features and experimental applications of the pZ expression system are presented:
1 IPTG-inducible expression of recombinant proteins
2 Tetracycline-inducible expression of recombinant proteins
2.1 Using Tetracycline, Anhydrotetracycline or Doxycycline as inducer
3 Graded induction of gene expression
3.1 Graded induction of genes controlled by P_A1lacO-1
3.2 Graded induction of genes controlled by P_lac/ara-1
3.3 Graded induction of genes controlled by P_LtetO-1 and P_N25tetO-1
4 Independent simultaneous and non-simultaneous expression of two gene activities
4.1 Coexpression of S. cerevisiae alpha-Glucosidase and E. coli chaperones DnaK, DnaJ: Influence on solubility and activity of alpha-Glucosidase
4.2 Simultaneous and non-simultaneous expression of two interacting MSP-1 subunits (p120 and p85) from P. falciparum: Influence on expression rates
5 Expression of toxic gene products: Production of restriction enzyme Cfr9I

1 IPTG-inducible expression of recombinant proteins

Experimental setup:
• Cultures of E. coli strain C600Zi harbouring pZE23p33, pZE23p190, pZE23luc, pZE23dnaK, or pZE23gst were grown to mid log phase in LB medium.
• Gene expression was induced by the addition of 1mM IPTG for 2.5 hrs.
• Proteins were separated by SDS-PAGE and stained with Coomassie.

Results
• Using pZE based vectors and IPTG inducible promoter P_A1lacO-1 proteins of various origins can be expressed to > 15% of total protein.
• No plasmid instabilities (variation in copy number, plasmid loss) were observed under any conditions of growth or long-term storage either in the presence or absence of selection antibiotic (data not shown).
• Recombinant protein levels strongly depend on posttranscriptional parameters, e.g. protein stability, mRNA stability, accessibility of translation initiation signal etc.

2 Tetracycline-inducible expression of recombinant proteins

Experimental setup:
• Cultures of DH5alphaZ1 harbouring pZE15dnaJ or pZE25groELES were grown to mid log phase in LB medium.
• Gene expression was induced by the addition of 200 ng/ml Anhydrotetracycline for 2.5 hrs.
• Proteins were separated by SDS-PAGE and stained with Coomassie.

Results:
• Using pZE based vectors and the Tetracycline inducible promoter P_N25tetO-1 proteins of various origin can be expressed to > 15% of total E. coli protein.
• No plasmid instabilities (variation in copy number, plasmid loss) were observed under any conditions of growth or long-term storage (data not shown).

2.1 Using Tetracycline (Tc), Anhydrotetracycline (aTc) or Doxycycline (Dox) as inducer

Experimental setup:
• Cultures of DH5alphaZ1 harbouring pZE15dnaJ or pZE25groELES were grown to mid log phase in LB medium.
• Gene expression was induced by the addition of either 200 ng/ml Tetracycline (Tc), Anhydrotetracycline (aTc) or Doxycycline (Dox) for 2.5 hrs.
• Proteins were separated by SDS-PAGE and stained with Coomassie

Results:
• Induction of expression can be achieved with different Tc derivatives.
• Levels of induction are highest if Doxycycline is used as inducer.
• Tc is not recommended for induction.
• The induction potential of aTc compares to Dox. Note: Long term storage of aTc in solution is not recommended.
• Dox is recommended for induction
• Induction by Dox is >1000 fold cheaper than induction with IPTG.

3 Graded induction of gene expression

3.1 Graded induction of genes controlled by P_A1lacO-1

Experimental setup:
• A culture of DH5alphaZ1 harbouring plasmid pZE13luc was grown to mid log phase in LB medium.
• After splitting the culture in multiple aliquots gene expression was induced by the addition of various concentrations of IPTG for 2.5 hrs and luciferase activity was determined.

Results:
• Expression levels of promoter P_A1lacO-1 correlate nearly linearly with IPTG concentrations. An expression plateau is reached above 0.1mM IPTG.

3.2 Graded induction of genes controlled by P_lac/ara-1

Experimental setup:
• A culture of DH5alphaZ1 harbouring plasmid pZE14luc was grown to mid log phase in LB medium. After splitting the culture in multiple aliquots gene expression was induced by the addition of various concentrations of IPTG and/or Arabinose for 2.5 hrs and luciferase activity was determined (A).

• A culture of DH5alphaZ1 harbouring plasmid pZE14grpE was grown to mid log phase in LB medium. After splitting the culture in multiple aliquots gene expression was induced by the addition of various concentrations of IPTG and/or Arabinose for 2.5 hrs. Bacteria were harvested and lysed, protein samples were separated by SDS-PAGE and stained with Coomassie (B).

Results:
• Expression levels of promoter P_lac/ara-1 correlate nearly linearly with IPTG concentrations between 0.005 and 0.1 mM.
• An expression plateau is reached above 0.1 mM IPTG.
• In the presence of 1 mM IPTG, expression levels correlate nearly linearly with Arabinose concentrations between 0.0005% and 0.02% (w/v).
• An expression plateau is reached above 0.05% Arabinose.

(B)

(A)

3.3 Graded induction of genes controlled by P_LtetO-1 and P_N25tetO-1

Experimental setup:
• A culture of DH5alphaZ1 harbouring pZE21luc was grown to mid log phase in LB medium.
• After splitting the culture in multiple aliquots gene expression was induced by the addition of various concentrations of Anhydrotetracycline for 2.5 hrs and luciferase activity was determined (A).

• A culture of DH5alphaZ1 harbouring plasmid pZA25dnaKJ_5grpE was grown to mid log phase in LB medium.
• After splitting the culture in multiple aliquots gene expression was induced by the addition of various concentrations of Anhydrotetracycline for 2.5 hrs.
• Proteins were separated by SDS-PAGE and stained with Coomassie (B).

Results:
• Expression levels of promoter P_LtetO-1 show sigmoidal character between 1 and 20ng/ml aTc.
• An expression plateau is reached above 20ng aTc.

(A)

(B)

4 Independent simultaneous and non-simultaneous expression of two gene activities

4.1 Coexpression of S. cerevisiae alpha-Glucosidase and E. coli chaperones DnaK, DnaJ: Influence on solubility and activity of alpha-Glucosidase

Experimental setup:
• Cultures of DH5alphaZ1 harbouring pZA35gluc and pZE14dnaKJ were grown to mid log phase in LB medium.
• Gene expression was induced with 200ng/ml Anhydrotetracycline (alpha-Glucosidase) and increasing concentrations of IPTG and Arabinose (dnaKJ) for 2.5 hrs.
• The soluble and insoluble protein fraction ("Supernatant" and "Pellet") was separated (A).
• Protein samples were separated by SDS-PAGE and stained with Coomassie, and alpha-Glucosidase activity was analyzed (B).

Results
• Yeast alpha-Glucosidase is almost completely insoluble and inactive if overexpressed in E. coli.
• Co-overexpression of DnaKJ increased alpha-Glucosidase solubility and activity up to 3 fold.
• DnaK and alpha-Glucosidase represent >10% of total cellular protein in the fully induced state (lanes 5 + 10).
• Note: DnaJ expression is lower than DnaK due to low translation initiation rates on bicistronic mRNA.

(A)

(B)

4.2 Simultaneous and non-simultaneous expression of two interacting MSP-1 subunits (p120 and p85) from P. falciparum: Influence on expression rates

Experimental setup:
• A culture of W3310Z1 harbouring pZA35p120 and pZE23p85 was grown to mid log phase in LB medium.
• After splitting the culture in aliquots gene expression was induced by:
i) 1 mM IPTG for 2.5 hrs
ii) 200 ng/ml Doxycycline for 2.5 hrs
iii) 1 mM IPTG and 200 ng/ml Doxycycline for 2.5 hrs
iv) 200 ng/ml Doxycycline for 2.5 hrs, followed by the addition of 1 mM IPTG for another 2.5 hrs.

Results:
• p120 and p85 show high expression levels if expressed separately.
• If gene expression is induced simultaneoulsy by IPTG and Dox, p120 is expressed to very low levels compared to p85 and compared to the single induction by Dox.
• p120 and p85 are expressed to nearly the same levels if p120 expression is induced 2.5 hrs before p85 is expressed.

5 Expression of toxic gene products: Production of restriction enzyme Cfr9I

Experimental setup:
_•_{Cultures of DH5alphaZ1 harbouring pZS*24cfr (- RBS), pZS*24cfr (+RBS), or pZA24cfr (+RBS) were grown to mid log phase in LB medium (Notes: Cfr9I was expressed without its cognate Methyltransferase M.Cfr9I. The presence of an efficient RBS (+ RBS) increased expression levels around 10 fold).}_{• Gene expression was induced by 1mM IPTG and 0.2% (w/v) Arabinose for 2.5 hrs.

•}_{Proteins were separated by SDS-PAGE and stained with Coomassie (A).}_{• Pulse-chase experiment}:
_{• A culture of DH5alphaZ1 harbouring pZA24cfr (+RBS) was grown to early log phase in minimal medium.

• After splitting the culture in aliquots gene expression was induced by 1mM IPTG and 0.2% (w/v) Arabinose.

_•_{Newly synthesized proteins were labeled at various time points by the addition of 35S-Methionine

•}_{Proteins were separated by SDS-PAGE and made visible by autoradiography (B)

Results:

• Cfr9I can be expressed to 2-3% of total cellular protein if encoded on a plasmid harbouring a p15A ori.

• No viable cells were detectable in any culture 20 min after induction of cfr9I (data not shown).}• No mucoid bacterial growth or plasmid instabilities were observed under conditions of repression (data not shown)._{• Expression of cfr9I leads to a decrease in protein production over time.

• 150 min after induction no protein systhesis can be detected (Note: expression of cfr9I causes immediate cell death).}}

_(A)

_(B)