methods for manipulating protein function

Question 1

in which ways can protein function be manipulated?

Answer 1

• DNA level
• mRNA level
• protein level

Question 2

what is transient transfection?

Answer 2

the process of introducing exogenous DNA into cells, but the plasmid DNA is not incorporated into the genome

Question 3

how can protein function be manipulated at DNA level?

Answer 3

• over-expression of wild type
• dominant active genes
• dominant negative genes

Question 4

how can protein function be modified at mRNA level?

Answer 4

RNA interference

Question 5

what are the steps for transient transfection?

Answer 5

1- mix plasmid with cationic lipids in an eppendorf tube, allowing a plasmid lipid complex to form
2- the complex is added to a Petri dish of exponentially growing cells, where it is taken into the cell via endocytosis
3- the complex is then incorporated into the nucleus where the membrane breaks down and reforms

Question 6

why is it ideal to generate a GFP tagged fusion protein with GFP present at the C-terminus?

Answer 6

the GFP protein is quite large and can interfere with protein folding if present at the N terminus

Question 7

how can protein function be modulated by phosphorylation?

Answer 7

the negative charge associated with phosphorylation can be mimicked by replacing serine or threonine with an amino acid that carries a n egative charge. this creates dominant active mutant that acts like it is constitutively phosphorylated

Question 8

what is site-directed mutagenesis?

Answer 8

an in vitro technique that allows you to selectively change one or more amino acids in a protein

Question 9

how has manipulation at RNA level been demonstrated?

Answer 9

• injection of mRNA that encodes Aquaporin 1 into xenopus eggs (which have no Aquaporin channels)
• cells lyse

Question 10

briefly outline RNA interference

Answer 10

• dsDNA activates the ribonucleoprotein complex (RISC)
• dsRNA binds dicer, which cuts RNA into 21 NT fragments which bind RISC
• argonaute 2 (AGO2) binds the fragments of RNA and selects the passenger strand for degradation
• the guide strand is left intact
• the guide strand directs AGO2 to complementary sequences, allowing these to be degraded

Question 11

how can RNA interference be demonstrated using fluorescent proteins?

Answer 11

• can design oligos where guide strand has homology to GFP or RFP
• when transfected, these cells do not express that particular protein

Question 12

how can CDK1/cyclin B demonstrate selective inhibition of protein function?

Answer 12

the protein is activated by forming the cyclin B/Cdk dimer. the cdk1 has an ATP binding site, which can be inactivated using an ATP competitor.. blocks mitosis