A general vector, pASK84, for cloning, bacterial production, and single-step purification of antibody Fab fragments.
The expression vector pASK84 was designed for the convenient cloning of immunoglobulin variable domain genes, as well as periplasmic secretion of the corresponding F(ab) fragment in Escherichia coli. The plasmid provides the constant domain genes of mouse IgG1/kappa with a hexa-histidine tag fused to the C terminus of the heavy chain. This strategy enables the rapid and efficient purification of the functional recombinant F(ab) fragment via immobilized metal affinity chromatography. The versatility of this expression and purification system is demonstrated using the variable domains of the well-characterized anti-lysozyme antibody D1.3.
Skerra
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Max-Planck-Institut für Biophysik, Frankfurt am Main, Germany.
D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli; D005803:Genes, Immunoglobulin / Q000235:genetics; D005822:Genetic Vectors; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification*
D007140:Immunoglobulin Fab Fragments; D011993:Recombinant Fusion Proteins