Question

Discussion questions Due at the beginning of next weeks' lab 1 How do expression vectors differ from vectors used for replicating DNA? (1 mark) 2 What is a fusion protein expression vector? What is the fusion component of pET28a? (2 mark) 3 Briefly discuss some potential problems in expressing eukaryotic proteins in E. coll. (2 marks) 4 Briefly describe a His-tag and how it interacts with its target. (2 mark) 5 How does imidazole act to elute the His-tagged protein from the chromatography column? (1 mark) 6 Why are small amounts of imidazole present in the equilibrium buffer and wash buffer? (1 mark) 7 What are inclusion bodies, and list two means by which their formation be avoided? (2 mark) 8 in your own words, provide a brief overview of next week's lab. Include the experimental objective, and how it integrates into the ultimate goal of expression and detection of GFP. (4 marks)

Answer 1

1)

Expression vectors contain elements not normally found in cloning vectors that allow the host cell to Transcribe and Translate the cloned fragment. These elements include, Promoter sequences, Start/Stop codons, Translation initiation sequences (Shine-Dalgarno sequence), etc.

2)

A fusion protein expression vector would contain a molecule like GFP or an N- or C- terminal tag like the His-tag sequence, positioned in such a way that both sequences are 'in-frame'. This means that both the pre-existing fusion protein sequence and the insert sequence are read in the same frame of triplet codons, and the protein produced has sequence identity with the protein found naturally.

pET28a has a 6x His tag which can be used as a fusion component.

3)

Eukaryotic proteins often require specialized chaperones for proper folding, which may not occur in prokaryotic cells. Additionally, post-translational modification of proteins differs between E. coli and Eukaryotes. Eukaryotic proteins synthesized in an E. coli host may not have the same post-translational modifications, affecting the activity of the protein.

4)

A His-tag is a sequence comprised of, usually, 6 Histidine residues. Histidine can bind Metal ions with high affinity, forming co-ordinate bonds with such atoms. When proteins with His-tags are purified, these proteins bind to columns with immobilized Nickel atoms, due to the interaction between the His-tag and the Nickle atoms.