Miscellaneous DNA Vector Systems

Highly efficient expression of recombinant proteins and very small peptides in E. coli

This new E. coli vector system is ideally suited for the stable and reproducible over-expression of proteins and - in particular - of polypeptides as small as five residues. The vector is a unique fusion construct based on the pET-3a expression system containing a novel sequence which promotes efficiently the expression of recombinant proteins as well as very small peptides in E. coli. Inherent in this novel sequence is an optimized affinity tag which allows for facile high-efficient affinity purification of the expressed product. A unique chemical cleavage site has also been engineered into this vector for easy removal of the fusion sequence from the target protein/peptide.

FEATURES:

• highly efficient expression of recombinant target proteins and very small polypeptides
• proteins which have proven to be toxic to E. coli are over-expressed only after induction and therefore do not interfere with cell growth
• the fusion peptide/protein as well as the final cleaved target protein/peptide can be rapidly purified from E. coli lysates using the same metal affinity chromatography column, which can be freshly charged with metal and reused many times
• a superior metal-chelating region exhibits stronger binding properties than the present commercially available Poly(His)-Tag, even in the presence of 3 M GuHCl (a mild denaturant which helps to increase the solubility of some proteins)
• this unique cloning system allows chemical release of the target peptide avoiding disadvantages associated with enzymic digestion of the target peptide
• enables easy and reproducible non-chemical synthesis of non-labelled peptides as well as uniformly labeling of peptides with ¹³C and/or ¹⁵N (for NMR structural and other investigations) at reasonable cost, and in adequate yield

• highly efficient expression of recombinant target proteins and very small polypeptides
• even toxic proteins are overexpressed after induction
• significantly higher affinity Poly(His)-Tag allows optimal purification of the fusion protein using metal affinity chromatography
• easy chemical release of the target peptide/proteincost-effective, time saving method to produce labeled and non-labeled peptides in adequate yields

(NOTE: The vectors pPEPTIDE and pPEPTIDE2 are identical except that the sequence encoding the highly expressed fusion protein is longer in pPEPTIDE2 (163 amino acids) than in pPEPTIDE (90 residues). Thus pPEPTIDE2 contains 5365base pairs.