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Flashcards in Protein engineering Deck (19)
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1

What are the 6 main library screening approaches?

Homologous probes
Heterologous probes
Oligonucleotide probes (designed from protein)
Subtractive hybridisation
Functional cloning
PCR

2

What problem does RACE transcription solve?

Allows amplication of the full transcript unlike RT-PCR, by includning the extreme 5' and 3' ends.

3

What is the key to protein engineering?

alteration of amino acid sequence

4

To get a gene that will be expressed:
1) Isolate ___ from tissue/___
2) Carry out _-PCR to produce _DNA
3) Use ___ to amplify ____ from _DNA pool
Use a proof reading ______
4) Check expected product size by _______
5) May need to ____ product from gel
6) Confirm sequence identity by _____
7) Subclone into _____ and _____ into bacterial cells.

To get a gene that will be expressed:
1) Isolate RNA from tissue/cell
2) Carry out RT-PCR to produce cDNA
3) Use PCR to amplify gene of interest from cDNA pool
Use a proof reading polymerase
4) Check expected product size by gel electrophoresis
5) May need topurify product from gel
6) Confirm sequence identity by sequencing
7) Subclone into vector and transform into bacterial cells.

5

Why is Bacillus subtilis sometimes used instead of E.coli?

Can be made to secrete the protein
Easier purification

6

What are important features of bacterial expression vectors that make them good hosts?

1) Selectable marker - eg drug resistance
2) Strong promoter, often inducible - e.g. b-galactosidase promotor stimulated by lactose
3) Contain the sequences needed for translation of the mRNA r.g. Ribosome binding site and ATG start codon
4) Contains suitable restriction sites e.g. multiple cloning site or polylinker

7

What are some problems with prokaryotic expression sytems? (4)

May not induce correct protein folding
Reducing environment
Protein insolubility
Proper post-translational modification may be difficult in host system.

8

Once expressed, how can we alter genes to produce modified proteins? (4)

Change specific amino acids (site-directed mutagenesis)
Have sections of polypeptide added
Have sections of polypeptide deleted (truncated)
Make hybrids of two proteins (fusion proteins)

9

What are peptide tags? What are they used for?

Polyhistidine or small motif recognised by an antibody.The fusion protein will bind to a nickel column for easy purification, being removed by histidine. Tag removed by a protease.

10

What are expression vectors?

They contain promoter sequences that are required for expression of the target gene

11

Give an example of a reporter protein, what are they used for?

GFP
Easily visible + allow the function of important gene regulatory signals to be studied (analyse the control of gene expression)

12

what do you need to ensure when ligation of insert into an expression vector

- May need to add restriction enzyme sites to insert DNA sequences
- must ensure the reading frame is maintained after re-ligation of insert into vector

13

What can changes can we make to engineered proteins?

Change specific amino acids
Add polypeptide sections
Delete polypeptide sequences
Create fusion proteins (hybrids)

14

What is site-directed mutagnesis, how is it better than chemical/radiation mutagenesis

- It changes the base sequence of the gene, producing the exact mutation you want, changing the amino acid in the protein.
- Chem/Rad muta is random, slow and inefficient

15

Conventional Site-Directed Mutagenesis:
1)A plasmid containing the gene to be altered is
__________________
2)A synthetic ________ with the required mutation hybridises to it (low stringency)
3)This _____ DNA synthesis in vitro, replicating the strand
4)Transfected into E.coli so that replication produces one _______ plasmid and one ______ plasmid

1)A plasmid containing the gene to be altered is separated into its two DNA strands
2)A synthetic oligomer with the required mutation hybridises to it (low stringency)
3)This primes DNA synthesis in vitro, replicating the strand
4)Transfected into E.coli so that replication produces one normal plasmid and one mutated plasmid

16

What are the problems with conventional site-directed mutagenesis methods?
What is the answer to this problem?

-Needs preperation of ssDNA
-Can be time consuming and difficult
-Multiple transformations needed since colonies express mutant and wild type

Answer is to introduce mutation into both strands using QuickChange

17

Site directed Mutagenesis: QuickChange
Performs thermal cycling to __________
Using ______, it incorporates mutation into _____ DNA strands.
Primers are extended and incorporated with ____ DNA polymerase
Template strands digested by ____: the recognition site contains a ______ base so only the _________ is digested.
Final product is _____ containing the mutation.
Transform cells and screen for uptake/mutation and ____ repair

Thermal cycling to denature the DNA template
Using 2 primers incorporates mutation into both DNA strands.
Primers are extended and incorporated with PluUltra DNA polymerase.
Template strands digested by Dpn1: the recognition site contains a methylated base so only the methylated, parent strand is digested.
Final product is dsDNA containing the mutation.
Transform cells and screen for uptake/mutation and nick repair.

18

Why should the primer be nearly, but not completely, complementary to it's template?

so that it hybridises under low stringency but still primes DNA synthesis

19

DnaD is a protein found in _______, and is involved in initiating ______.
It consists of an N-terminal domain, which mediates _______, and a C-terminal domain, which _______.

DnaD is a protein found in Bacillus subtilis, and is involved in initiating DNA replication.
It consists of an N-terminal domain, which mediates oligomerisation, and a C-terminal domain, which binds DNA.