IBA : Protein Tools

Overview
pASK-IBAplus vectors
pASK-IBA (Strep-tag vectors)
pASK-IBA (6xHistidine-tag and double-tag vectors)
pPR-IBA vectors (Strep-tag vectors)
pPR-IBA vectors (One-STrEP-tag vectors)
For individual maps please refer to the data sheets
Sequences can be downloaded here
All vectors at a glance can be found here

Overview of Strep-tag^®vectors
For expression in E. coli we are offering a large variety of pASK-IBA vectors allowing the expression of recombinant proteins carrying Strep-tag. Except for the antibiotic resistance genes (Amp^R, Cam^R) the pASK-IBA vectors differ only between the XbaI and HindIII restriction sites. The differences are demonstrated in the map below: you can choose between N- or C-terminal tag, periplasmic or cytoplasmic expression and/or endoproteinase cleavage sites for the removal of the tag (factor Xa, enterokinase, thrombin and now also TEV protease). Please note, that usually the tag does not have to be removed.

pASK-IBAplus vectors
The cytoplasmic pASK-IBA vectors, which are already well established for expression of Strep-tag^® and/or 6xHistidine-tag fusion proteins, are now offered as "plus" version. This new version contains an improved translation initiation site increasing primary protein yield while the remaining sequence of the "plus" vectors (e.g. pASK-IBA3plus) is identical to its "non-plus" predecessor (e.g. pASK-IBA3*). The "non-plus" version is from now on only available on request.
Below please find a complete list of pASK-IBA E. coli vectors allowing the expression of recombinant proteins carrying Strep-tag and/or 6xHistidine-tag.

Strep-tag^®vectors

**Overview of double-tag and 6xHistidine-tag* vectors**

In addition to Strep-tag^® vectors, we are also offering double-tag vectors with both, Strep-tag and 6xHistidine-tag, as well as 6xHistidine-tag vectors. Two different affinity tags on a protein yield highest purification factors and allow purification of full-length proteins under denaturing or physiological conditions. Also, purification protocols directly from the culture medium can be optimized.
Vectors differ only in between the XbaI and HindIII restriction sites.

6xHistidine-tag and double-tag vectors

*Exception: pASK-IBA43plus contains a NheI restriction site between the ATG start
codon and the N-terminal 6xHistidine-tag, which is not included in pASK-IBA43.

Structure of a selection of pASK-IBA expression vectors with Strep-tag or Strep-tag and 6xHistidine-tag. pASK-IBA2, pASK-IBA4 and pASK-IBA6 are also available with chloramphenicol resistance instead of ampicillin resistance. MCS, f1, Amp^R, ori, t_lpp , ompA, Xa, thb, ent, and tetR denote the multiple cloning site, intergenic region of phage f1, b-lactamase gene, origin of replication of the pUC family of plasmids, lpp terminator of transcription, ompA signal sequence, factor Xa recognition sequence, thrombin recognition sequence, enterokinase recognition sequence and tet repressor gene, respectively. The expression cassette is under transcriptional control of the tetA promoter/operator. The tet repressor gene has been placed as a second cistron immediately behind the cistron encoding b-lactamase and is thus under transcriptional control of the constitutive b-lactamase promoter.
Digesting the vector with one of the type IIS restriction enzymes BsaI, Eco31I, or BsmFI leads to the generation of defined 5' overhangs in the upstream and downstream elements, which are not mutually compatible and can therefore not re-ligate. Upstream elements may be DNA sequences encoding the OmpA signal peptide, an initiator methionine, Strep-tag II, 6xHistidine-tag or the protease recognition sequence, while downstream elements may be the Strep-tag II, 6xHistidine-tag or a stop codon. The gene to be cloned must be equipped with compatible overhangs, e.g. by incorporation of appropriate restriction sites by PCR and subsequent cleavage.

References:
Skerra A, Schmidt TGM, 2000: Meth. Enzymol. 326: 271-304. Use of the Strep-tag and streptavidin for recombinant protein purification and detection.
Skerra A, 1994: Gene 151: 131-135. Use of the tetracycline promoter for the tightly regulated production of a murine
antibody fragment in Escherichia coli.

pPR-IBA vectors (T7 promoter)

The primers created for cloning into pPR-IBA1 and pPR-IBA2 are also compatible with our pASK-IBA expression vectors:

pPR-IBA1 cloning primers are suitable for pASK-IBA3 and pASK-IBA33
pPR-IBA2 cloning primers are suitable for pASK-IBA4, pASK-IBA5 and pASK-IBA35

Sequencing primers are described here.

E.coli: Strep-tag® and/or 6xHistidine-tag vectors

Overview of double-tag and 6xHistidine-tag* vectors

pPR-IBA vectors (T7 promoter)

E.coli: Strep-tag^® and/or 6xHistidine-tag vectors

**Overview of double-tag and 6xHistidine-tag* vectors**