Cloning of ACP-tag Fusions in pACP-tag(m) (N9135)

Protocol

  1. Cloning by PCR
    To subclone an appropriate cell surface signal peptide into pACP-tag(m) to create a N-Terminal fusion to the ACP-tag, use the available restriction sites SacI , SacII , NotI , EcoRV or HindIII which are located upstream of the ACP-tag. 

    To subclone the gene of interest into pACP-tag(m) to create a C-Terminal fusion to the ACP-tag use the available restriction sites downstream of the ACP-tag: SbfI (PstI ), AscI , BamHI , XhoI , ApaI and KpnI

    Note: When making a C-Terminal fusion to the ACP-tag, note that there is a stop codon between the BamHI and XhoI sites, so SbfI (PstI), AscI, or BamHI must be used as the 5´ cloning site for your insert.

    Primer Design and Cloning Hints:

    • Design your PCR primers to include a sufficient overlap with the sequence of the gene you want to amplify.
    • You may also want to include a stop codon 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) as a fusion with the ACPwt gene, the resulting plasmid can be used for transient expression of the ACP-tag fusion proteins in a suitable cell line.
    • NEB 10-beta Competent E. coli (High Efficiency) (NEB #C3019) is recommended for propagating and subcloning this vector.
  2. 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 BamHI and XhoI sites, so SbfI (PstI), AscI, or BamHI must be used as the 5´ cloning site for your insert.