Abstract
Monoclonal antibodies which recognize the cell surface-exposed part of outer membrane protein PhoE of Escherichia coli were used to select for antigenic mutants producing an altered PhoE protein. The selection procedure was based on the antibody-dependent bactericidal action of the complement system. Using two distinct PhoE-specific monoclonal antibodies, seven independent mutants with an altered PhoE protein were isolated. Among these seven mutants, five distinct binding patterns were observed with a panel of 10 monoclonal antibodies. DNA sequence analysis revealed the following substitutions in the 330-residue-long PhoE protein: Arg-201----His (three isolates), Arg-201----Cys, Gly-238----Ser, Gly-275----Ser and Gly-275----Asp. It is argued that amino acid residues 201, 238, and 275 are most likely directly involved in antibody binding and, therefore, exposed at the cell surface. Together with Arg-158, which was previously shown to be cell surface exposed as it is changed in phage TC45-resistant phoE mutants, these four positions show a remarkably regular spacing, being approximately 40 residues apart. A model is suggested in which the PhoE polypeptide repeatedly traverses the outer membrane in an antiparallel beta-pleated sheet structure, exposing eight areas to the outside which are all separated by approximately 40 residues.
Highlights
From the Departmentof Molecular Cell Biologyand Institutefor Molecular BioZugy, State University of Utrecht, Transitorium 3, Paduahn 8,3584 CH Utrecht, The Netherlands
In thepresent study,we have shown that monoclonal antibodies (mAbs) can be used to select antigenic mutants of E. coli that produce an altered outer membrane PhoE protein
The selection procedure was based on the antibody-dependent bactericidal action of the complement system
Summary
A Results arebased on ELISA experiments andi,n a number of cases, on cell immunoradioassays. ++, wild-type level; +, 10-30% of wild-type level; -, no binding detected
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