349845 Method of Using Fastcloning Technique to Express Thymidylate Synthase with Polyhistidine-Tag to Aide in the Extraction of a Pure Protein
Method of Using FastCloning Technique to Express Thymidylate Synthase with Polyhistidine-tag to Aide in the Extraction of a Pure Protein
Catherine Suchanek, Thelma Abeysinghe and Amnon Kohen
Department of Chemistry, The University of Iowa, Iowa City, IA 52242, USA
FastCloning is a PCR cloning method used to insert DNA sequences into a plasmid vector or gene. Using three main steps, the FastCloning technique is much quicker and more economical than traditional PCR cloning methods. Recent studies using the FastCloning method to transform a vector and insert into competent Escherichia Coli cells suggest that the procedure is highly effective and reproducible. By adopting the FastCloning method, the objective is to combine a polyhisitidine-tag (His-tag) with the gene for wild type thymidylate synthase (Thy A). Ultimately, so that the protein with the his-tag can be transformed to competent cells and the pure protein may be extracted using a nickel (Ni2+) column.
An insert containing the Thy A gene, and a vector (pET28 plasmid vector) containing the gene for the His-tag, are amplified separately using the FastCloning PCR method. PCR products for both the insert and vector are confirmed using an agarose gel run. Both PCR products are digested with Dpn1 to rid the mixture of the parent DNA strands. The insert and vector are then transformed into E. Coli XL 10 Blue cells, which are later cultured and run through SDS page as well as DNA sequencing to confirm that the product is Thy A with His-tag. Upon confirmation, the culture is run through a Ni2+ column to remove any proteins without the His-tag and washed with imidazole to extract the pure protein with the His-tag.
Work thus far has reached confirmation of the PCR products for both the vector and the insert. Future work includes increased trials for confirmation of transformation products using SDS page and DNA sequencing, as well as work with the nickel column to ensure that the pure protein may be obtained.
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