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Gene recovery. Fluorescence activated cell sorting (FACS). Yeast cell surface display. Directed Evolution. Mutagenesis. Labeling.
Fluorescence activated cell sorting (FACS)
Yeast cell surface display
Creation of New Chondroitin Lyases Through Novel Methods Andrew Harper*, Mark J. OlsenDepartment of Mathematics, Chemistry, and PhysicsWest Texas A & M University Canyon, TX 79015
Chondroitin sulfates are natural polysaccharides found in the extracellular matrix. They are not only altered during tumor progression and metastasis but also directly affect wound healing, immunological responses, stem cell development, stem cell maturation, neurological development, and neuron growth. Chondroitin sulfates are divided into disaccharide subunit classifications based on differences in sulfation and epimerization. Each disaccharide subunit consists of an N-acetyl-galactosamine residue and a uronate residue and is given a letter designation, as seen in the table. The use of different subunits is thought to carry information and several studies have shown that subunit differences affect protein recognition of these polysaccharides.
Chondroitin ABC lyase I (chondroitinase ABC or cABC) from Proteus vulgaris is an enzyme capable of cleaving many different kinds of these subunits, apparently through the use of a necessary substrate binding domain. It is a very useful and promising enzyme, but has various flaws depending on the intended application. For example, cABC has already been very successful in improving the rate and degree of recovery from spinal and central nervous system injury in mice, even though it has poor thermostability and is known to have low activity against chondroitin D subunits, which are important in various neurological processes. Chondroitin ABC lyase I is one of several chondroitin lyases useful in chondroitin sulfate analysis, and while it is useful in some situations due to its broad substrate selectivity, there is a great need for more selective chondroitin lyases for analytical purposes. For example, there is no known chondroitin lyase that selectively cuts previously mentioned chondroitin D subunits. Given the known strengths of cABC, it appears to be a likely predecessor for the creation of a more powerful generation of analytical and therapeutic enzymes. A simple, elegant, and direct approach to the engineering of this protein is to control the selectivity of the substrate binding/presentation domain through directed evolution.
The nucleotide sequence coding for the n-terminal domain (i.e. the substrate binding domain) of cABC has been displayed on the surface of the yeast using the vector pCTCON. Mutant libraries of the protein will be created using error-prone PCR. Yeast cells will be fluorescently labeled to determine the substrate binding affinity and level of protein expression. Fluorescently labeled peptides will be conjugated to chondroitin sulfates of known subunits such that the labeled polypeptides will be bound with the substrate to the n-terminal domain. This means that the more tightly a protein binds a specific chondroitin type, the more fluorophores will be bound to the cell expressing that protein, causing the cell to appear more fluorescent at that wavelength. Protein expression will be determined using a fluorescent, antibody-based system integrated into the vector. Cells with the desired fluorescence (and hence the desired protein expression and substrate binding affinity) will be retrieved from the library using a fluorescence activated cell sorter (FACS). The nucleotide sequence of the altered n-terminal domain will be retrieved from these cells and sequenced. After determining the effect of the changes once the domain has been reintegrated into the whole enzyme, the sequence may be used as the template for the next generation of libraries or may be considered a successful end point.
Chondroitin sulfates are sugars known to carry information through their sulfation patterns. This plays important roles in many processes, such as cancer and stem cell differentiation. A major hurdle to deciphering and using this information is the lack of specific enzymes available to cut these sugars. This project will attempt to make a generalist enzyme (chondroitinase ABC I) that cuts many types of chondroitin subunits into a specialist enzyme that will cut only the specific, rare, and important chondroitin D subunit. The substrate selectivity of the entire enzyme will be changed only by altering the substrate binding domain. By using this method, this project will answer questions about the effect of substrate binding on selectivity as well as providing new tools for studying such sugars. This is a novel application of the established technique of directed evolution. A library of substrate binding domain mutants will be made by introducing random point mutations at a controlled rate. Changes in enzyme activity will be measured by display on yeast cell surfaces and measuring changes in cell fluorescence as the enzyme cuts chondroitin sulfates attached to fluorescent compounds which then adhere to the cell surface.
Above left: Chondroitin ABC lyase I from Proteus vulgarisAbove right: The substrate binding domain from chondroitin ABC lyase I
Below: The cycle of protein evolution through directed evolution using random mutagenesis and fluorescent activated cell sorting
Results, Conclusions, & Future Directions
The chondroitin lyase ABC I gene has been extracted from P. vulgaris and the n-terminal domain has been integrated into the yeast cell surface display vector. The next steps are to verify protein expression and substrate binding. After that, we will begin generation of libraries. The entire chondroitinase ABC gene has been already been inserted into the pCTCON vector, and in the future will be engineered for thermostability.
Killgore Research Enhancement Grant
USDA Seed Grant
Dr. James Hallmark