Martin, Sophie E. V.; (1994) The expression and metal affinity purification of single-chain antibodies. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Single-chain antibody fragments (ScFv's) which were originally derived from the anticancer antibody B72.3, were genetically altered in order to facilitate their purification by immobilized metal affinity chromatography (IMAC). Metal-binding tails comprising two or five histidine residues were incorporated, by site-directed mutagenesis, into the carboxyl terminus of the antibody fragments. In addition, two of the four tails included a region of five residues derived from the hinge sequence of the original B72.3 whole antibody. The four configurations were designated ScFv(his)2, ScPvhinge(his)2, ScPv(his)5 and ScPvhinge(his)5 according to the number of histidine residues and the presence or absence of the hinge region. Supernatants from E.coli cells expressing ScFv constructs with a hinge region, contained more total protein and more antibody fragment than those lacking the hinge residues. The ScFv variants were systematically purified on immobilized copper, nickel and zinc ion adsorbents. Different elution methods were also investigated. The highest yields were achieved by purifying ScFvhinge(his)5 on zinc(II)-IDA-agarose and eluting either with an increasing imidazole concentration or a decreasing pH gradient. Using pH elution, ScFvhinge(his)5 was eluted at pH 5.8 and purified 56-fold from the E.coli supernatant to a purity of 77%. ScFvhinge(his)5 bound more strongly to nickel(II)-IDA- agarose and was eluted at pH 4.8. Although the yield from nickel(II)-IDA purifications was lower, the purity of eluted antibody fragment was higher. Copper(II)-IDA-agarose was deemed unsuitable for purifications because of its high affinity for other proteins present in the supernatant. Copper ion leaching occurred when the supernatant was applied to the column, but this was minimised by dialysis before loading. The metal-binding tail with five consecutive histidines had the highest affinity for metals. ScFv(his)2 did not bind the IMAC column, nor could it be purified successfully by antigen affinity chromatography. This indicated that it was incorrectly folded. ScFvhinge(his)2 bound nickel(II)-IDA-agarose weakly and was eluted isocratically at pH8 which did not enable a complete separation from contaminating E.coli proteins. ScFv(his)5, required a lower pH for elution, but its original low concentration in the supernatant hindered its investigation. The pentahistidine tail, with the hinge region, is the tail of choice for purifications of single-chain antibodies.

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