Partially structured state of the functional VH domain of the mouse anti‐ferritin antibody F11

Abstract

An antibody combining site generally involves the two variable domains, VH from the heavy and VL from the light chain. We expressed the individual VH domain of the mouse antihuman ferritin monoclonal antibody F11. The loss of affinity was not dramatic (K a = 4.0U U10 7 M 31 versus 8.6U U10 8 M 31 for the parent antibody) and comparable to that previously observed for other VHs. However, the functional VH domain adopted a partially structured state with a significant amount of distorted secondary and compact yet greatly destabilized tertiary structures, as demonstrated by spectroscopic and calorimetric probes. These data provide the first description for a functional antibody domain that meets all the criteria of a partially structured state. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Expression of anti-tumor recombinant IgG- and IgE-like genes in eukaryotic cells

“…During the past few years, new approaches for recombinant antibody design have been developed, with particular emphasis on enhancement of their affinity and stability, as well as on the delay of antibody excretion from the organism. Earlier we have reported the production of genes encoding new, functionally active anticancer antibodies expressing in bacteria [2,17,18] and plant cells [19]. The fundamentally different approach is the design of genetic constructs capable for the expression of full-length recombinant antibodies intended for target in situ (in the organism) recognition.…”

“…Protein transfer from gel to membrane (Millipore) was performed according to manufacturer's protocol. One membrane was stained with anti-His alkaline phosphatase-conjugated Abs [17], while the other one was visualized with an anti-4D5 rabbit antibody obtained in our laboratory.…”

Camelized Rabbit-derived VH Single-domain Intrabodies Against Vif Strongly Neutralize HIV-1 Infectivity

“…A similar decrease in the relative antigen-binding affinity was demonstrated in previous studies when isolated VH antibody fragments were compared with their parent antibodies. 45,46 In contrast, control experiments Antibody fragments were extracted from periplasm as described in Materials and Methods. The pellets (insoluble fraction) and the periplasm (soluble fraction) were analyzed by an SDS-PAGE 15% gel, followed by Western blot using the HRP-conjugated anti-HA monoclonal antibody.…”