Metal affinity engineering of proinsulin carrying genetically attached (His)10-X-Met affinity tail and removal of the tag by cyanogen bromide.
An E. coli expression clone coding for human proinsulin, which was fused to NH2-terminal beta-galactosidase, was engineered for the separation from host proteins by introducing peptide devices, and for the sequential removal of the fused polypeptide by cyanogen bromide in front of the NH2 terminal residue (methionine) of the human proinsulin gene. Short synthetic genes encoding oligopeptide residues including (Glu)n, (His)n, (Trp)n, and (Ser)n (n = 10 or 11), which have certain characteristic physical properties such as metal-affinity, polarity, hydrophobicity, and hydrophilicity, respectively, were inserted at the junction region of the gene fusion. Interestingly, it was found that among the oligopeptides, the oligohistidine residue as an affinity-tag has greatly facilitated the procedures for FPI purification, particularly in the manner of selective metal-affinity precipitation. The chelating peptide covering the NH2-terminal beta-galactosidase portion could then be removed simply after purification to generate a protein with the natural amino acid sequence of proinsulin by cyanogen bromide.
Ko
J H
JH
Molecular Biology Laboratory, Genetic Engineering Research Institute, K.I.S.T., Taejon, Korea.
Chung
W J
WJ
Koh
S
S
Park
B C
BC
Kwon
S T
ST
Kim
C H
CH
Lee
D S
DS
D000345:Affinity Labels; D000595:Amino Acid Sequence; D001483:Base Sequence; D011232:Chemical Precipitation; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular; D003488:Cyanogen Bromide / Q000737:chemistry*; D005818:Genetic Engineering / Q000379:methods*; D006639:Histidine / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D008670:Metals; D008715:Methionine / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry* / Q000235:genetics*; D011384:Proinsulin / Q000235:genetics* / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013431:Sulfates / Q000737:chemistry / Q000302:isolation & purification
D000345:Affinity Labels; D008670:Metals; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D013431:Sulfates; D006639:Histidine; D011384:Proinsulin; D008715:Methionine; D003488:Cyanogen Bromide