Biomolecular engineering of human protein Galectin-2: effects on ligand binding, stability and physical state

Chen, Xiaojing (2013). Biomolecular engineering of human protein Galectin-2: effects on ligand binding, stability and physical state PhD Thesis, Aust Institute for Bioengineering & Nanotechnology, The University of Queensland.

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Author Chen, Xiaojing
Thesis Title Biomolecular engineering of human protein Galectin-2: effects on ligand binding, stability and physical state
Formatted title
Biomolecular Engineering of Human Protein Galectin-2: Effects on Ligand Binding, Stability and Physical State
School, Centre or Institute Aust Institute for Bioengineering & Nanotechnology
Institution The University of Queensland
Publication date 2013
Thesis type PhD Thesis
Supervisor Anton Middelberg
Lizhong He
Total pages 179
Total colour pages 11
Total black and white pages 168
Language eng
Subjects 030403 Characterisation of Biological Macromolecules
100403 Medical Molecular Engineering of Nucleic Acids and Proteins
100302 Bioprocessing, Bioproduction and Bioproducts
Formatted abstract
Recombinant protein therapeutics are drawing more and more attention in preventing and curing diseases, and they have been used in a wide range of clinical practices. In spite of that, biopharmaceuticals often possess several intrinsic limitations that hinder their medical applications. These shortcomings typically result in a short circulation half-life, undesired immune response and poor stability that reduce their therapeutic functions and clinical potencies dramatically. To date, many approaches have been developed to modify biopharmaceuticals with the aim of overcoming these mentioned limitations, as well as to create novel modes of action with increased therapeutic value. However, the rational design and engineering of proteins is always a knowledge-based process. The modification of a protein can be a long process and often includes more than one optimization cycle in which achieved knowledge and a new engineering strategy alternate, until the desired product can be created. Hence, this study aims to deliver comprehensive knowledge required for rational design of protein therapeutics with novel and desired properties, including a tuneable bioactivity, an improved stability and a prolonged in vivo circulation lifetime. A potential pharmaceutical protein, human galectin-2 (hGal-2) was used as a proof-of-principle model in this work due to its similarity to other carbohydrate-binding proteins. This thesis firstly reports a bioprocess for producing deuterated recombinant protein with high yield and purity. Both upstream processing and downstream purification methods have been successfully developed to support advanced structural studies, also supplying practical knowledge of deuterium modification. Secondly, this project systematically investigated the consequent impact on protein bioactivity and physical state generated by modification using site-directed mutagenesis (SDM) and site-specific PEGylation. The connections between affinity and the physical state of hGal-2 have also been revealed. Thirdly, this work thoroughly examined protein stability as engineered outcomes. It demonstrated that the thermodynamic stability of hGal-2 was significantly improved by both SDM and PEGylation, but was weakened by deuteration. It also described influences brought by external factors including ligand binding and buffer environment. Fourthly, this thesis provided important conformational information of PEGylated protein conjugate by using methods of small-angle scattering (SAS). The fundamental knowledge obtained from this study can be applied to guide engineering strategies and promote better designs of therapeutic proteins in order to obtain desired functionalities and optimized biophysical properties. It will eventually contribute to the development of new and safer protein-based medicines with enhanced patient compliance.
Keyword Recombinant therapeutic protein
Human galectin-2
Ligand binding
Physical state
Site-directed mutagenesis

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Created: Tue, 08 Oct 2013, 16:28:34 EST by Xiaojing Chen on behalf of Scholarly Communication and Digitisation Service