PCR cloning - Recommendations

  • For an optimal performance of a new protein it is recommended to test different E. coli strains and E. coli expression vectors either induced by a temperature shift or by IPTG.
  • With respect to the purification procedure we would recommend introducing a “tag” at the N- or C-terminal end of the recombinant protein. There are E. coli expressions vectors available containing different “tags”. If the recombinant protein is used mainly in in vitro experiments (e.g. cell culture studies) we recommend for example a “His-tag” (6x Histidin) or a “Strep-tag II” (8 amino acids) [www.iba-go.de]. The “tag” is normally too small to interfere with the activity of the protein. For “Strep-tag II” the possibility exists to create an authentic protein by cleavage of the tag. If the recombinant protein is needed for animal studies (e.g. mouse, rat) we recommend the use of an “Fc-tag” (about 26 kDa) to increase the stability and half-life of the protein in the circulation. As an “Fc-tag” ReliaTech can offer human IgG1 and the mouse IgG2b fragments.