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  • Cloning of ACP-tag Fusions in pACP-tag(m)-2 (N9322)

    Introduction

    Cloning by PCR
    To subclone a gene of interest or an appropriate cell surface signal peptide into pACP-tag(m)-2 to create an N-terminal fusion to the ACP-tag, use the available restriction sites: NheI, EcoRV(blunt), AscI, SwaI (blunt), BsrGI, AgeI or EcoRI which are located upstream of the ACP-tag.
    To subclone the gene of interest into pACP-tag(m)-2 to create a C-terminal fusion to the ACP-tag, use the available restriction sites downstream of the ACP-tag: SbfI, BamHI, PmeI(blunt), XhoI, PacI and NotI.

    Note: When making a C-terminal fusion to the ACP-tag, note that there is a stop codon between the PacI and NotI sites, so SbfI, BamHI, PmeI, XhoI or PacI must be used as the 5´ cloning site for the insert.

    PmeI and XhoI cannot be used together for cloning because they share a cytosine as part of their recognition sequences.

    Primer Design and Cloning Considerations:

    • Design the PCR primers to include a sufficient overlap (15–20 bp) with the sequence of the gene to be amplified.
    • For fusion to the C-terminus of the ACP-tag, a stop codon may be included at the C-terminus of the fusion (in front of the downstream cloning site) in order to terminate translation at that position.
    • For fusions upstream of the ACP-tag, ensure that a start codon is included. The addition of a Kozak sequence (e.g. GCCRCCATG, where the start codon is underlined) will increase the translation efficiency.
    • In general, any linker peptide between the proteins should be kept short to avoid degradation by proteases. If required, specific protease cleavage sites can be introduced into the linker peptide.
    • Care should be taken to design the cloning so that the fusion partners in the resulting construct are in frame.
    • Perform the PCR reaction and subsequent cloning steps according to established protocols for molecular biology.
    • After subcloning the gene of interest into pACP-tag(m)-2 as a fusion with the ACPwt gene, the resulting plasmid can be used for stable or transient expression of the ACP-tag fusion proteins in a suitable cell line.
    Direct Cloning
    Direct cloning can also be used to make fusions with the ACP-tag. This is only possible if the fusion partner has compatible sites adjacent to the gene of interest. 

    Care should be taken to design the cloning so that the fusion partners in the resulting construct are in frame.

    Note: When making a C-terminal fusion to the ACP-tag, note that there is a stop codon between the PacI and NotI sites, so SbfI, BamHI, PmeI, XhoI or PacI must be used as the 5´ cloning site for the insert. 

    PmeI and XhoI cannot be used together for cloning because they share a cytosine as part of their recognition sequences.

    Troubleshooting 

    Cloning of the Gene of Interest 

    If subcloning of the gene of interest with the ACP-tag does not work, reconfirm all the cloning steps (primer design, choice of restriction site, etc.). If all steps are confirmed as being correct, then try the cloning using different restriction sites. Be sure to include a positive and negative control for the ligation reaction.

    Alternatively, try to subclone the ACP-tag gene into an expression vector already containing the gene of interest.

    Protocol

    1. Thaw a tube of Lemo21(DE3) Competent E. coli cells on ice for 10 minutes.
    2. Add 1–5 µl containing 1 pg–100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4–5 times to mix cells and DNA. Do not vortex.
    3. Place the mixture on ice for 30 minutes. Do not mix.
    4. Heat shock at exactly 42°C for exactly 10 seconds. Do not mix.
    5. Place on ice for 5 minutes. Do not mix.
    6. Pipette 950 µl of room temperature LB or SOB into the mixture.
    7. Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
    8. Warm selection plates to 37°C.
    9. Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in LB or SOB.