Unit 6

A _______________ protein is a protein that contains multiple protein/polypeptide sequences that are __________________ attached to form one protein product.

Common reasons for making fusion proteins (Advantages):

1) One protein can be used to tag another protein for ______________ (e.g. attaching GST) through affinity chromatography.
2) One protein can be used to tag another protein for ___________ (e.g. attaching GFP).
3) One protein can make another protein more STABLE/SOLUBLE– allow for purification and biochemical analysis.

Disadvantages of fusing two proteins:

1) Fusion may result in altered __________ - this will negatively impact function.
2) Fusion can change the transport __________ within the protein. (gets taken to the wrong place)
3) Fusion changes the IMMUNOLOGICAL properties of the protein - antibodies will recognize both parts of a fusion protein.

The reading frame is determined by the first start ________ the ribosome encounters.

Base insertions or deletions that result in a shift of the reading frame are termed _____________

An insertion that is a multiple of three bases _____(will/will not) result in a frameshift

How do you adjust reading frame?

You can design ___________ to amplify and digest the insert, insert 1 or 2 nucleotides between the restriction endonuclease site and the gene sequence to shift the frame. This will help us create a PCR product with the correct reading frame for our vector.

When do we need to consider reading frame?

1) Whenever we are inserting a gene into a vector.
2) When we are N-terminally tagging a protein.
3) When we are C-terminally tagging a protein.
4) Either B or C.
5) None of the above