Protein Engineering

Question 1

What two main ways do proteins evolve?

Answer 1

a. gradual accumulation of beneficial/enhancing mutation

b. recombinartion gives chimeras with enhanced properties (genetic events or CRISPR)

Question 2

Why might you want to use protein engineering? Give an example

Answer 2

To see how a protein works, or to alter the way it works. For example, you might make to want toxins that bind to different Rs in an insect to overcome toxin resistance

Question 3

Comapre Site-Directed Mutagenesis with Random Mutsagenesis

Answer 3

SDM:

• need knowledge of the protein’s structure/genetic sequence to design PCR primers
• uses PCR with primer-oligonucleotide pairs carring desired mutation (indel) –> induces SNP
• can be used to test function of a specific gene or induce desired changes, eg silencing a START codon –> genetic knockdown of tumour prodcuing cell

RM:
- different methods:

1. oligonucleotide-directed mutagenesis using a defenerate primer - so you have some control over the mutation but you don’t know which one will take
2. Gene propegation cloned in plasminds passed
   through a mutator strain, eg bacteria XL1-Red. Strains such as this one have defected repair pathways; XL1-Red has muatation in the mutD gene, making the mistmatch repair pathway defective, and also mutated mutT is defective in repairing oxidative DNA damages. Thus this strain introdcues mutations at v high rate.
   Pros: quick, easy, repeating the DNA replication round as many times as you like to get more mutations, no hazardous mutagens or cacinogens required. Also 5,000 fold higher rate of spontaneous mutation than E.Coli
   USed in PACE
3. USe an error-prone PCR polymerase such as Taq Polymerase which doesnt correct errors! alternatively increase Mg in the PCR reaction mixture.
   - all can be used to make small changes gradually, for example seeing which genes are beneficial and which are essential for breeding crops

Question 4

What is DNA shuffling? Why is it useful?

Answer 4

Using maternal and paternal DNA strands to combine randomly and make a new, mutant strand.

Temperature changes cause fragmentation and reanealling - sections of the gene from each parent which are the same (high sequence identity) will allign and reanneal.

Can do up to 50 cycles of assembily then use PCR to amplify full length sequences.

Question 5

What is surface phage display, and why do you need it? Give an example of how this has been applied.

Answer 5

Protein engineering methods might produce 10000s if mutants and you want to identify the ones that are revelant/beneficial.
You could use screening with E/Coli but this is labourious, so instead use PACE (relies on surface display of phages (MIII protein) - selecting for an interaction between insect Cahedrin R and the Bt Toxin - Cry1Ac). Interacton means that the MII gene is transcribed into the MIII protein, allowing for phage surface display and infection of new cells. If there is no interaction, there wil be no reininfection (thus no propegation of mutations). Ecoli was used as host cell, with the accessory plasmid containing the 2-Hybrid System (Cahedrin R), promotor and M3III gene. When the Selection phage inserts the phage genome into the cell, there will either be an interacton between this Cry1A toxin or there will not. The interaction depends on the mutatgenic plasmid transcribing mutated versions of the phage genome.

PACE was used to decrease insect resistance to toxins, by allowing the toxins to bind to new Rs. It is a useful method as it can be done in vivo - thus selecting for both mutations and essential gene maintenence in one. For example, the first mutations made the toxins instable in the insect gut (so they were actually less toxic!!). But they use illumina to identify and then used site-directed mutagenesis to fix this. This is why it is essential to do PACE in vivo if you can, to select for the environmental pressures.

Question 6

What reasons might cause mutagenically evolved toxinis to not be active towards the insect?

Answer 6

1. instable in the gut –> broken down by enzymes thus not active anymore
2. mutations might prevent the toxin from destroying the cells it targets, ie not able to form a pore
3. the toxin might be binding to the wrong part of the Cahedrin R, or there has been a conformational change in the R or the phage?