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" Engineering novel selectivity into metalloantibodies and targeting PcrV "
Farokhi, Elinaz
Huxford, Tom
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Latin Dissertation
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English
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| Record Number
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905520
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TL5wm7c7b3
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| Main Entry
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Farokhi, Elinaz
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| Title & Author
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Engineering novel selectivity into metalloantibodies and targeting PcrV\ Farokhi, ElinazHuxford, Tom
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| College
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UC San Diego
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| Date
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2020
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2020
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| Abstract
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Antibodies are a class of proteins that are secreted from our immune system to neutralize pathogens. They are very specific to the target cells and stably folded therefore they have become engaging for many scientists for decades. We were interested to study a novel group of antibodies that interacts to metals. Our findings indicated that metals are effective on stabilizing the antigen: antibody interaction. Previously, we were able to crystallize LT1009 antibody which is a humanized version of murine LT1002 IgG antibody. The crystal structure indicated that LT1009 employs two bridging calcium ions in the binding site to antigen. The biologically active lipid sphingosine-1-phosphate (S1P) was the antigen. Flame atomic absorption spectroscopy (FAAS) confirmed that murine LT1002 also binds calcium in solution and inductively coupled plasma-mass spectrometry (ICP-MS) indicates that, although calcium is the preferred metal binding cofactor, LT1002 can also bind to magnesium and, to a much lesser extent, barium. Isothermal titration calorimetry revealed that LT1002 binds two calcium ions endothermically with dissociation constants in the high μM range. Finally, an engineered, recombinantly expressed and purified antibody Fab fragment consisting of the murine germ-line encoded light and heavy chain genes from which LT1002 is likely derived and exhibits calcium binding profile as the mature antibody.Furthermore, we were interested to apply other metal bindings to the precursor metalloantibody. Therefore, we designed a large scale expression method to express the precursor antibody in Sf9 cells. Then, we were interested to design a plasmid by using site directed mutagenesis to change the key residues that were involved with the binding sites of Ca2+ and antigen. After that we were able to express and purify mutant antibody but this time the new metalloantibody binds to Zn2+. Engineered zinc antibodies can have the potential for site specific conjugation. Moreover, Zn2+ can protect cysteine from oxidation at the binding site. We propose that LT1002 is representative of a class of naturally occurring metalloantibodies that are evolutionarily conserved within the genomes of mammalian species.
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Huxford, Tom
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UC San Diego
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