Extending linker sequences between antigen-recognition modules provides more effective production of bispecific nanoantibodies in the periplasma of E. coli

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Abstract

The use of technology for the production of single-domain antibodies (NANOBODY® molecules, also referred to as nanoantibodies, nAb, or molecules based on other stable protein structures) and their derivatives to solve current problems in biomedicine is becoming increasingly popular. Indeed, the format of one small, highly soluble protein with a stable structure, fully functional in terms of specific recognition, is very convenient as a module for creating multivalent, bi-/oligo-specific genetically engineered targeting molecules and structures. The production of nAb in the periplasm of the E. coli bacterium is a very convenient and fairly universal way to obtain analytical quantities of nAb for the initial study of the properties of these molecules and the selection of the most promising nAb options. The situation is more complicated with the production of bi- and multivalent derivatives of initially selected nAbs under the same conditions. In this work, extended linker sequences (52 and 86 aa) between antigen-recognition modules in cloned expression constructs were developed and applied in order to increase the efficiency of production of bispecific nanoantibodies (bsNB) in the periplasm of E. coli bacteria. Three variants of model bsNBs described in this study were produced in the periplasm of bacteria and isolated in soluble form with preservation of the functionality of all protein domains. If earlier our attempts to produce bsNB in the periplasm with traditional linkers no longer than 30 aa were unsuccessful, the extended linkers used here provided a significantly more efficient production of bsNB, comparable in efficiency to the traditional production of the original monomeric nAbs. The use of highly elongated linkers can presumably be useful for increasing the efficiency of production of other bsNBs and similar molecules in the periplasm of E. coli bacteria.

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About the authors

S. V. Tillib

Institute of Gene Biology, Russian Academy of Sciences; Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Author for correspondence.
Email: tillib@genebiology.ru
Russian Federation, 119334, Moscow; 119991, Moscow

O. S. Goryainova

Institute of Gene Biology, Russian Academy of Sciences; Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: tillib@genebiology.ru
Russian Federation, 119334, Moscow; 119991, Moscow

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Supplementary files

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2. Fig. 1. The scheme of the expression structure used for the synthesis of bsNT and the sequence of linkers separating the sequences encoding nAT. From left to right, the design indicates: the lactose promoter site (Plac/operator); the ribosome binding site (RBS); the beginning of translation (arrow); the signal peptide for periplasmic localization (pelB); the sequences encoding nAT (VHH1 and VHH2) separated by a linker sequence (the amino acid sequences of the used linkers are indicated at the bottom drawing); HA-tag sequences (YPYDVPDYA) and six histidine residues at the very end. ILZ is a trimerizing domain, "isoleucine lightning"

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3. Fig. 2. SDS-PAGE and enzyme immunoassay of bsNT aBet-l1-aICAM1 and aICAM1-l1-aBet. a is the electrophoretic separation in a 5-19% gradient SDS polyacrylamide gel of proteins from periplasmic extracts (1, 2) and affine purified NATs (3, 4). The arrow shows the position of the bsNT. There is a marker (M) in the leftmost track and the dimensions of the marker strips are indicated (in kDa). b – Immunoassay of binding of the obtained bsNT at a concentration of 0.4 mcg/ml with immobilized recombinant antigens. The averaged data of three dimensions are presented, indicating the area of data dispersion

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4. Fig. 3. SDS-PAGE and enzyme immunoassay of bsNT aHA-l2-E7(aIgA), also referred to as (HN-E7)x3. a – Electrophoretic separation in a 5-19% gradient SDS polyacrylamide gel of affine purified bsNT, initial (1), overshoot on a filter with a nominal molecular cutoff a mass of 100 kDa (2), purified from fine impurities and, presumably, trimerized bsNT (3), delayed above the filter with a molecular weight cutoff of 100 kDa. The arrow shows the position of the monomeric bsNT. A marker (M) is applied in the rightmost track and the dimensions of the marker strips are indicated (in kDa). b – Immunoassay of binding of the obtained bsNT (HN-E7)x3, as well as control monovalent NATs (E7 – against IgA and HN13 – against hemagglutinin HA-H5 avian influenza virus) at a concentration of 1 mcg/ml with immobilized recombinant antigens (IgA and HA-H5). The control wells were without antigen (columns "without AG"). The averaged data of three dimensions are presented, indicating the area of data dispersion

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