BIOC 300D1 Lecture Notes - Lecture 3: Eif4E, Elution, Eif4Ebp1

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Translation: mrna proteins, process mediated by ribosome and other factors, in most cases, methionine is the first amino acid incorporated in the nascent polypeptide, met encoded by aug in eukaryotes and prokaryotes. Although gug and auu encode valine and isoleucine respectively, they can also encode methionine if preceded by the shine-dalgarno sequence in prokaryotes only. It directs the binding of the ribosome at the 5" end so it can start scanning for a translation initiation site. Inducible promoter: 2 proteins are expressed in pgex for this experiment, gst, gst-4ebp fusion protein. Extraction from cells: after expression, need to extract total protein from e. coli cells, methods: Lysozyme: disrupts polysaccharide linkages in bacterial cell wall peptidoglycan (found in tears and saliva) Dtt: disrupts disulfide linkages, prevents protein folding. Extraction from cells: done by your tas. Resuspended the cells in pbs (physiological buffer) Added dtt to reduce non-native disulfide linkages. Centrifuged to bring down pellet of cellular debris.

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