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Single-Molecule Analysis by Biological Nanopores
Published in Shuo Huang, Single-Molecule Tools for Bioanalysis, 2022
An alternative means of preparing biological nanopores is by prokaryotic expression followed with purification by fast protein liquid chromatography. In this approach, a tag protein such as His-tag [54] or Strep-tag [36] is normally placed on either terminus of the target protein for later purification purposes. The prokaryotic protein expression system can efficiently produce several mg of protein in 3–4 days. However, if the target protein is cytotoxic to the host cell during expression, difficulties may ensue. This method also requires more complicated purification procedures to eliminate interferences from the background proteins generated by the host cell. Site-directed mutagenesis is sometimes needed to optimize the performance of the protein nanopore [55]. This can be accomplished by direct synthesis of the mutated gene or with a site-directed mutagenesis kit.
Tailoring of recombinant FDH: effect of histidine tag location on solubility and catalytic properties of Chaetomium thermophilum formate dehydrogenase (CtFDH)
Published in Preparative Biochemistry & Biotechnology, 2019
Hacer Esen, Saadet Alpdağtaş, Mehmet Mervan Çakar, Barış Binay
The production of recombinant proteins is essential for biotechnological applications. These proteins should be highly expressed and purified for being used and beneficial in research. Many recombinant proteins can be modified by the addition of a specific sequence (or tag) to the N- or C-terminus of the protein for purification.[1] The glutathione S-transferase, the FLAG-tag, the maltose-binding protein, the Strep-tag, the S-tag, thioredoxin, and His-tags are some of the known tags.[2] Among them, His-tags are widely used because of their low molecular weight.[3]