Improving Yields of Mammalian Proteins in Bacterial Systems
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Date
2021-05
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The Ohio State University
Abstract
Proteins are a growing class of drugs due to the extensive therapeutic potential they possess. Proteins found in living systems can be modified to become effective therapeutics for a variety of diseases such as cancer or an infection. Engineering proteins allows us to modify these proteins in a way that they are not only more effective, but also safe for use.
Designing stable proteins with desired biophysical properties can be challenging. Mutations made can create undesirable and unpredictable changes to the protein. Not only that, but factors such as disulfide bonds and protease activity during expression can make it difficult to express proteins efficiently. Here I present work on removing disulfide bonds in order to increase the therapeutic potential of antibody fragments and work on expressing specific mammalian proteins in bacteria.
Antibodies have become an important point of study for cancer therapeutics. Antibody fragments are able to detect and bind to specific antigens and can be used as a therapeutic or diagnostic tool. One type of antibody fragment is single-chain variable fragments (scFV). These are the smallest functional unit of an antibody formed by fusing the variable heavy (VH) and the variable light (VL) domains with a peptide chain (linker). Expressing these in E. coli can be difficult due to their reliance on disulfide bonds for stability. Proteins with disulfide bonds need to be expressed in an oxidizing environment
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so that they are able to form. In E. coli this means that proteins must be sent to the periplasm instead of being expressed in the cytosol, which can cause decreased expression of the protein. This issue can be solved in one of two ways: either by removing the disulfide bonds without losing protein structure or by using a specialized E. coli that are able to easily express proteins with disulfide bonds.
Using a specialized strain of E. coli with an oxidizing cytosolic environment, Shuffle T7 Express, we were able to express scFV in E. coli without needing to send it to the periplasm. ScFV expressed in the cytosol remained soluble in the monomeric state, whereas scFV that took on higher oligomeric states became insoluble and would be found in the pellet after cells were lysed. We also used Rosetta to help design potential scFV candidates that have their disulfide bonds removed. By creating scFV with no disulfide bonds, we can create antibody fragments with better therapeutic potential and express large quantities in the cytosol of E. coli with a standard reducing environment in the cytosol.
Caspase-3 is a type of cystine-aspartic acid protease that is essential in the process of apoptosis. Caspase 3 is an active protease expressed in mammals that specifically cleaves D-X-X-D sequences. This protein is difficult to express in E. coli in its active form since its protease activity will cleave random proteins in E. coli, making it toxic and potentially lethal to the cell. By cloning rational variants of active caspase-3 protein, I was able to diagnose issues in the expression that can be helpful in being able to express large sums of caspase-3 in bacterial systems.
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Keywords
Protein Engineering, Antibody Fragments, Caspase-3