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MCDB121LREcombinantProteinsEcoli - Expression and...

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Expression and Purification of Recombinant Proteins in E. Coli Jharrett Carrillo, MCDB 121La, Yale University Abstract The various components of this laboratory exercise were meant to cultivate an understanding of E. Coli and its usefulness in the study of recombinant DNA technology. The expression of eukaryotic proteins can be studied in E. Coli as it is a model organism for the study of various processes such as transcription, translation, gene regulation, and mechanisms of mutation. The objective of this lab was to clone a gene encoding the protein of interest, lysing the cells to extract the contents, and isolating the protein by the use of an affinity column. An epitope was used as a marker for the protein of interests, making them has an overall negative charge. The proteins were then run down a nickel affinity column which has an overall positive charge. The proteins were removed from the column using imidazol and then sorted by size via an SDS gel in order to separate the proteins of interest from the endogenous proteins. The gel was submerged in a Colde Blue Stain solution and stained for an hour. The findings of this exercise suggest that E. Coli are ideal for expressing recombinant proteins, as determined by the observation of the gel. Introduction Escherichia coli is a model organism for observing processes ranging from transcription, translation, gene regulation, to mechanisms for mutation. They are relatively inexpensive to grow and have rapid biomass accumulation due to its short life cycle. E. Coli is valuable for the study of the expression of complex proteins due to the many tools available to facilitate gene cloning and expression in this bacterium. The objective of this
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