16 research outputs found
Anchoring of proteins to lactic acid bacteria
The anchoring of proteins to the cell surface of lactic acid bacteria (LAB) using genetic techniques is an exciting and emerging research area that holds great promise for a wide variety of biotechnological applications. This paper reviews five different types of anchoring domains that have been explored for their efficiency in attaching hybrid proteins to the cell membrane or cell wall of LAB. The most exploited anchoring regions are those with the LPXTG box that bind the proteins in a covalent way to the cell wall. In recent years, two new modes of cell wall protein anchoring have been studied and these may provide new approaches in surface display. The important progress that is being made with cell surface display of chimaeric proteins in the areas of vaccine development and enzyme- or whole-cell immobilisation is highlighted.
Ribosome Display Selection of a Murine IgG1 Fab Binding Affibody Molecule Allowing Species Selective Recovery Of Monoclonal Antibodies
Affinity reagents recognizing constant parts of antibody molecules are invaluable tools in immunotechnology applications, including purification, immobilization, and detection of immunoglobulins. In this article, murine IgG1, the primary isotype of monoclonal antibodies (mAbs) was used as target for selection of novel binders from a combinatorial ribosome display (RD) library of 1011 affibody molecules. Four rounds of selection using three different mouse IgG1 mAbs as alternating targets resulted in the identification of binders with broad mIgG1 recognition and dissociation constants (KD) in the low nanomolar to low micromolar range. For one of the binders, denoted Zmab25, competition in binding to full length mIgG1 by a streptococcal protein G (SPG) fragment and selective affinity capture of mouse IgG1 Fab fragments after papain cleavage of a full mAb suggest that an epitope functionally overlapping with the SPG-binding site in the CH1 domain of mouse IgG1 had been addressed. Interestingly, biosensor-based binding experiments showed that neither human IgG1 nor bovine Ig, the latter present in fetal bovine serum (FBS) was recognized by Zmab25. This selective binding profile towards murine IgG1 was successfully exploited in species selective recovery of two different mouse mAbs from complex samples containing FBS, resembling a hybridoma culture supernatant
Surface display of a functional single-chain Fv antibody on staphylococci.
Two different host-vector expression systems designed for cell surface display of chimeric receptors on Staphylococcus xylosus and Staphylococcus carnosus have been evaluated for surface display of a mouse immunoglobulin G1(kappa) [IgG1(kappa)] anti-human IgE single-chain Fv (scFv) antibody fragment. To achieve surface anchoring of the chimeric receptors containing the scFv, the cell surface attachment regions from Staphylococcus aureus protein A were used in both expression systems. The different chimeric receptors could be recovered from cell wall extracts of both S. xylosus and S. carnosus, and surface localization was demonstrated by taking advantage of a serum albumin-binding reporter region present within the two types of receptors. In addition, the two different recombinant staphylococci carrying hybrid receptors containing the scFv were demonstrated to react with the antigen, which was human IgE, in whole-cell enzyme-linked immunosorbent assays. This is the first report of an antibody fragment expressed in a functional form anchored to the surface of gram-positive bacteria. The potential use of recombinant gram-positive bacteria as whole-cell diagnostic devices or alternatives to filamentous phages for surface display of scFv libraries is discussed