Purification of recombinant proteins by chemical removal of the affinity tag.
The efficient removal of a N- or C-terminal purification tag from a fusion protein is necessary to obtain a protein in a pure and active form, ready for use in human or animal medicine. Current techniques based on enzymatic cleavage are expensive and result in the presence of additional amino acids at either end of the proteins, as well as contaminating proteases in the preparation. Here we evaluate an alternative method to the one-step affinity/protease purification process for large-scale purification. It is based upon the cyanogen bromide (CNBr) cleavage at a single methionine placed in between a histidine tag and a Plasmodium falciparum antigen. The C-terminal segment of the circumsporozoite polypeptide was expressed as a fusion protein with a histidine tag in Escherichia coli purified by Ni-NAT agarose column chromatography and subsequently cleaved by CNBr to obtain a polypeptide without any extraneous amino acids derived from the cleavage site or from the affinity purification tag. Thus, a recombinant protein is produced without the need for further purification, demonstrating that CNBr cleavage is a precise, efficient, and low-cost alternative to enzymatic digestion, and can be applied to large-scale preparations of recombinant proteins.
Rais-Beghdadi
C
C
Institut de Biochimie, Université de Lausanne, Epalinges, Switzerland.
Roggero
M A
MA
Fasel
N
N
Reymond
C D
CD
D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity; D003488:Cyanogen Bromide / Q000737:chemistry*; D004926:Escherichia coli; D006639:Histidine / Q000737:chemistry*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010963:Plasmodium falciparum; D015800:Protozoan Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification
D015800:Protozoan Proteins; D011994:Recombinant Proteins; C041595:circumsporozoite protein, Protozoan; D006639:Histidine; D003488:Cyanogen Bromide