Site Directed Mutagenesis and Protein Engineering

Question 1

Name the aromatic amino acids.

Answer 1

1. Tryptophan (Trp)
2. Phenylalanine (Pry)
3. Tyrosine (Tyr)

Question 2

What is a mutation?

Answer 2

a change in the nucleic acid sequence (DNA) of an organism’s genetic material

Question 3

Define directed mutagenesis

Answer 3

a change in the nucleic acid sequence (or genetic material) of an organism at a specific predetermined location

Question 4

Describe protein engineering.

Answer 4

Protein engineering utilizes genetic manipulation to change the coding sequence and by extension modify the properties of the associated protein.

Question 5

__________ and ________ are used to change/modify the properties of a protein.

Answer 5

Site-directed mutagenesis and protein engineering

Question 6

What are the properties of the protein that would want to be changed?

Answer 6

Michaelis constant (Km)
Vmax
Thermal stability
pH stability
Cofactor requirement
Specificity
Sensitivity

Question 7

What is the Km of an enzyme?

Answer 7

the tightness of the substrate binding to the enzyme.

Question 8

What is the Vmax of an enzyme?

Answer 8

the maximal rate of conversion of the substrate to the products.

Question 9

How does modifying the protein affect the Km and Vmax?

Answer 9

Km - increases the specificity of the reaction and reduces side reactions
Vmax - increases the number of products produced.

Question 10

How does modifying the proteins affect the following: a) pH or thermal stability
b) cofactor
c)specificity

Answer 10

a) an increase may allow the protein to be used under conditions where it would typically be denatured.
b) abolishing this may be beneficial under certain industrial conditions and hence save money
c) increase in this will decrease the prevalence of undesirable side reactions.

Question 11

It may be possible to modify an existing protein to produce an altered protein via what?

Answer 11

1. Directed mutagenesis
2. Protein engineering

Question 12

Why do we modify the gene instead of the protein?

Answer 12

When the gene is modified, the host organism continues to make the altered protein every time translation occurs.

Question 13

Why is it not feasible to modify the protein?

Answer 13

If the protein is modified, each time the protein is produced by the organism it would have to be modified.

• Also chemical modification is harsh, non-specific and is a repetitive process

Question 14

What components do all the different procedures of directed mutagenesis have in common/utilized?

Answer 14

1. A synthetic oligonucleotide
2. A vector (a plasmid or M13)

Question 15

What type of mutation is made with site-directed mutagenesis?

Answer 15

Point mutation

Question 16

What are the two must-knows for site-directed mutagenesis?

Answer 16

1. the precise nucleotide sequence in the region of DNA that encodes the messenger RNA (mRNA) codon that is to be changed
2. the amino acid changes that are being introduced.

Question 17

Describe how site-directed mutagenesis would occur in the M13 phage (theoretically).

Answer 17

1. the cloned gene was inserted into the double-stranded form of an M13 bacteriophage vector
2. The recombinant vector’s single-stranded form (M13 plus-strand) was isolated and mixed with a synthetic oligonucleotide. This oligonucleotide was precisely complementary to a segment of the cloned gene except for one nucleotide- the targetted one for a change
3. The oligonucleotide hybridizes to the complementary region of the cloned gene if it is added in an amount much in excess of that of the M13 DNA
4. The 3′ end of the hybridized oligonucleotide acts as a primer site for the initiation of DNA synthesis that uses the intact M13 strand as the template
5. The replication is catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I. T4 DNA ligase is added to ensure that the last nucleotide of the synthesized strand is joined to the 5′ end of the primer.
6. Transformation into E.coli
7. Theoretically, because plasmid DNA is replicated semiconservatively, half of the phage that are formed carry the wild-type sequence and the other half contain the mutated sequence that has the specified nucleotide change. Phage produced in the initial transformation step are propagated in E. coli, and particles that contain only the mutated
   gene are identified by DNA hybridization under highly stringent conditions.
8. Original nucleotide acts as a probe to identify the mutated gene. ds of M13 identified and mutated gene is exised by restriction enzymes.

Question 18

Theoretically, site-directed mutagenesis is supposed to produce a 50/50 yield of wild type to mutated genes but that doesn’t happen. What does happen?

Answer 18

the expected 50% of the M13 viruses carrying the mutated form of the target gene are not recovered. Rather, for a variety of technical reasons, only
around 1% of the plaques actually contain phage carrying the mutated gene.

Question 19

What has been done to increase the yield of site-directed mutagenesis with M13?

Answer 19

1. introduce the M13 viral vector carrying the gene that is to be mutagenized into an E. coli strain that has two defective enzymes of DNA metabolism - a defective form of dUTPase (dut) and a defective uracil-N-glycosylase (ung).
2. Cells without a functional dUTPase have an elevated intracellular level of dUTP, which in turn causes a few dUTP residues to be incorporated into DNA during replication instead of dTTP.
3. In the absence of functional uracil N-glycosylase, the dUTP residues that were spuriously incorporated into DNA cannot be
   removed.
4. the ung gene degrades the original strand of the M13, leaving behind the mutated strand.