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View / Download 7.8 Mb Sortase As a Tool in Biotechnology and Medicine by Joseph J. Bellucci III Department of Biomedical Engineering Duke University Date:_______________________ Approved: ___________________________ Ashutosh Chilkoti, Supervisor ___________________________ Charles A. Gersbach ___________________________ Kris Wood ___________________________ Fan Yuan ___________________________ Michael R. Zalutsky Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biomedical Engineering in the Graduate School of Duke University 2016 i v ABSTRACT Sortase As a Tool in Biotechnology and Medicine by Joseph J. Bellucci III Department of Biomedical Engineering Duke University Date:_______________________ Approved: ___________________________ Ashutosh Chilkoti, Supervisor ___________________________ Charles A. Gersbach ___________________________ Kris Wood ___________________________ Fan Yuan ___________________________ Michael R. Zalutsky An abstract of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biomedical Engineering in the Graduate School of Duke University 2016 i v Copyright by Joseph J. Bellucci III 2016 Abstract We have harnessed two reactions catalyzed by the enzyme sortase A and applied them to generate new methods for the purification and site-selective modification of recombinant protein therapeutics. We utilized native peptide ligation —a well-known function of sortase A— to attach a small molecule drug specifically to the carboxy-terminus of a recombinant protein. By combining this reaction with the unique phase behavior of elastin-like polypeptides, we developed a protocol that produces homogenously-labeled protein- small molecule conjugates using only centrifugation. The same reaction can be used to produce unmodified therapeutic proteins simply by substituting a single reactant. The isolated proteins or protein-small molecule conjugates do not have any exogenous purification tags, eliminating the potential influence of these tags on bioactivity. Because both unmodified and modified proteins are produced by a general process that is the same for any protein of interest and does not require any chromatography, the time, effort, and cost associated with protein purification and modification is greatly reduced. We also developed an innovative and unique method that attaches a tunable number of drug molecules to any recombinant protein of interest in a site-specific manner. Although the ability of sortase A to carry out native peptide ligation is widely used, we demonstrated that Sortase A is also capable of attaching small molecules to iv proteins through an isopeptide bond at lysine side chains within a unique amino acid sequence. This reaction —isopeptide ligation— is a new site-specific conjugation method that is orthogonal to all available protein-small conjugation technologies and is the first site-specific conjugation method that attaches the payload to lysine residues. We show that isopeptide ligation can be applied broadly to peptides, proteins, and antibodies using a variety of small molecule cargoes to efficiently generate stable conjugates. We thoroughly assessed the site-selectivity of this reaction using a variety of analytical methods and showed that in many cases the reaction is site-specific for lysines in flexible, disordered regions of the substrate proteins. Finally, we showed that isopeptide ligation can be used to create clinically-relevant antibody-drug conjugates that have potent cytotoxicity towards cancerous cells. v Contents Abstract ......................................................................................................................................... iv List of Tables ................................................................................................................................. xi List of Figures .............................................................................................................................. xii Acknowledgements ................................................................................................................. xvii 1. Introduction ............................................................................................................................... 1 1.1 Proteins and antibodies for cancer therapy .................................................................. 1 1.1.1 Antibody-drug conjugates ......................................................................................... 3 1.1.2 Protein-polymer conjugates ....................................................................................... 4 1.2 Challenges associated with therapeutic protein production ...................................... 5 1.2.1 Column chromatography ........................................................................................... 5 1.2.2 Purification tags ........................................................................................................... 8 1.2.3 Elastin-like polypeptides .......................................................................................... 10 1.3 Challenges associated with protein modification ...................................................... 11 1.3.1 Non-specific conjugation methods introduce heterogeneity .............................. 11 1.3.2 Available methods for modifying proteins ........................................................... 13 1.4 Sortase A catalyzes a unique reaction that can be applied to protein purification and modification ................................................................................................................... 20 2. Chromatography-free purification and site-specific labeling of recombinant proteins using sortase A ............................................................................................................................ 22 2.1 Introduction ..................................................................................................................... 22 2.2 Materials and methods .................................................................................................. 23 vi 2.2.1 Molecular biology used to construct fusion proteins ........................................... 23 2.2.2 Expression and purification of fusion proteins ..................................................... 24 2.2.3 Sortase reactions ........................................................................................................ 26 2.2.4 Characterization of purified target proteins .......................................................... 27 2.2.5 TNFα and TRAIL cytotoxicity assays ..................................................................... 29 2.3 Results and discussion ................................................................................................... 30 2.3.1 Binary fusion reaction ............................................................................................... 31 2.3.2 Ternary fusion reaction............................................................................................. 39 2.3.3 Bioactivity of the purified proteins ......................................................................... 40 2.3.4 Simultaneous tag removal and site-specific conjugation of small molecules to the carboxy-termini of substrate proteins ....................................................................... 42 2.3.5 TRAIL-ELP fusions show potent in vitro and in vivo cytotoxicity towards TRAIL-sensitive cell lines .................................................................................................. 48 2.4 Results and discussion ................................................................................................... 54 3. A non-canonical function of sortase enables site-specific conjugation of small molecules to lysine residues in proteins .................................................................................. 55 3.1 Introduction ..................................................................................................................... 55 3.2 Materials and Methods .................................................................................................. 58 3.2.1 Protein expression and purification ........................................................................ 58 3.2.2 Protein-small molecule conjugation reactions ...................................................... 58 3.2.3 LC-MS/MS and biotin:protein molar ratio ............................................................. 59 3.2.4 Immunofluorescence microscopy ........................................................................... 60 3.3 Results and Discussion .................................................................................................. 61 vii 3.3.1 Proof-of-concept of sortase-catalyzed isopeptide ligation using peptides ....... 61 3.3.2 Sortase-catalyzed protein polymerization through isopeptide bonds .............. 63 3.3.3 Conjugation of small molecules to proteins using sortase-catalyzed isopeptide ligation ................................................................................................................................. 65 3.3.4 Isopeptide ligation of multiple small molecules to a single protein ................. 70 3.3.5 Analysis of the isopeptide ligation product by tandem mass spectrometry .... 78 3.3.6 Protein-small molecule conjugates retain bioactivity..........................................
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