1. The DNA sequence of the GFP gene near the 3\' end of the ORF is given below.

Question

1. The DNA sequence of the GFP gene near the 3' end of the ORF is given below. Using a codon chart, show in concept how you could generate a fusion of the ER-retention signal KDEL (Lys-Asp-Glu-Leu) to the C-terminus of GFP. Assume the ORF reading frame is as given. ...CAC AAG CTT AAT TGA

2. The above molecular "construct" provides a mechanism to retain GFP in the ER. What additional molecular manipulation must be done to ensure the encoded GFP protein is targeted to the ER in the first place? Be as specific as you can.

Explanation / Answer

1. KDEL- a sequence of four amino acids, located at the extreme carboxyl terminus of proteins, acts as a signal for localization of proteins to the lumen of ER.

Fusion of a KDEL sequence to carboxyl terminus of lysozyme results in its retention in the ER

Addition of KDEL sequnce to cathepsin, a lysosomal enzyme results in localization in the ER.

2. Proteins with c-terminal KDEL sequence can be captured in the Cis-golgi or an intermediate compartment and returned to ER. KDEL containing protein bind to the receptor in an acidic post ER compartment. KDEL receptors is involved in tetrograde transport of its cargo to ER.

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