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234 Arnau et al.
Fig. 2. The pQE-1 vector for N-terminal histidine tag (His-tag) constructions to
facilitate removal of N-terminal residues using TAGZyme. Restriction sites within the
multiple cloning sites, DNA sequences and the corresponding N-terminal amino acid
sequences are shown. DAPase cleaves off dipeptides from the N terminus, and DAPase
digestion stops at the glutamine residue (Q) in the presence of excess Qcyclase. See
http://www.qiagen.com for more information about pQE vectors.
2. Materials
2.1. Molecular Biology: Cloning Strategy to Incorporate the UZ-HT15
His-Tag Sequence as a Universal Tag
The coding sequence of interest can be amplified using a primer that includes
the His-tag adjacent to the target gene sequence and provides a restriction site
for cloning in any vector that contains a, for example, NcoI site (C_CATGG)
or a BspHI site (T_CATGA) or a PciI site (A_CATGT) at the start codon. This
design provides a good translation start in E. coli. It is also important that the
amino acid sequence starts with MK to ensure a high expression level and an
effective cleavage with DAPase ((5), see also Subheading 1).
General primer design for UZ-HT15 His-tag sequence with added Gln stop
(67 nucleotides) (see Note 2).
MetLysHisGlnHisGlnHisGlnHisGlnHisGlnHisGlnGln
NNNNTCATGAAACACCAACACCAACATCAACATCAACATCAACATCAACAG...18 bp
overlap (target gene)
Cloning vectors for use with TAGZyme are also available. The Gln DAPase
stop point for DAPase can be introduced by cloning the protein coding sequence
into TAGZyme vector pQE-2 (5). Here, any uneven amino acid position can