1
Q
Aims of Protein Engineering
A
- probe mechanism
- create novel proteins
- improve catalytic function (turnover/binding affinity)
- alter specificity
- improve stability
2
Q
Plasmid Based Mutagenesis
A
- gene in plasmid with mutation target site
- design primers complementary to mutation site and surrounding regions
- denature plasmid and anneal mutation primers
- incorporate primers resulting in nicked circular strands
- digest non mutated template with RE
- transform circular nicked DNA into cells which repair nicks in mutated plasmid
3
Q
Overlap Extension Methods
A
- two primers—one for each end—are used per sequence.
- To splice two DNA molecules, special primers are used at the ends that are to be joined.
- For each molecule, the primer at the end to be joined is constructed such that it has a 5’ overhang complementary to the end of the other molecule. - Following annealing when replication occurs, the DNA is extended by a new sequence that is complementary to the molecule it is to be joined to.
- Once both DNA molecules are extended in such a manner, they are mixed and a PCR is carried out with only the primers for the far ends.
- The overlapping complementary sequences introduced will serve as primers and the two sequences will be fused
- no cut sites needed
4
Q
Alanine Scanning Mutagenesis
A
- systematic approach to engineering
- used to investigate role of residues
- obtain a stable construct
- every residue mutated to Alanine to test outcome
- functional assay assesses effects
5
Q
Random mutagenesis
A
- chemical mutagenesis
- plasmids chemically treated inducing damage
- gene amplified by PCR and cloned - PCR approaches
- sloppy PCR
- increased error rate to increase mutations (0.6-2%) - Plasmid in E. Coli
- treat with chemicals or rays to induce mutations and propagate in repair defective E. Coli strains - DNA shuffling
- in vitro recombination
- cut randomly mutated variants and purify
- use as PCR template and fragments assemble into longer novel gene forms
6
Q
DNA Shuffling with LacZ alpha
A
- DNA fragment with lacZa amplified via PCR
- digested fragments reassembled into full length gene at high concentration
- average size of product increases with each cycle
- cloning into plasmid yielded many blue colonies reflecting mutations occuring during reassembly
7
Q
Iterative process of mutation
A
- isolate wild type gene
- mutation of gene
- expression of mutant gene
- selection of functional mutant enzyme5. isolation of genes for improved enzymes
- repeat
8
Q
Variant Screening
A
- essential for random variants
- standard substrate conversion
- fluorogenic surrogate substrate
- coupled consecutive signalling reaction
- altered substrate may not represent real life
9
Q
Iterative Saturation mutagenesis
A
- rapid directed evolution
- mutating in very specific areas
- combination of targeted and random
- generate cell site variants and scan for best to further mutate
10
Q
Bacillus subtiliis
A
- example of iterative saturation mutagenesis
- mutated to be made more thermostable (less flexible)
- mutated residues with high B factors determined via crystallography
- iterative mutation was used to show which variants were most stable based on melting curves
11
Q
Engineering UapA
A
- uric acid xanthine transporter from Aspergillus nidulans
- high affinity high capacity hydrogen ion symporter
- antifungal drug target
- 14 TM domains with 2 half helices containing binding site
12
Q
Expression of UapA
A
- express in S. cerevisiae yeast as GFP fusion to show membrane trafficking
- functional assay to measure activity and expression
13
Q
Stability of UapA
A
- SEC for separation
- highly a helical protein
- long term stability trial with purified protein incubated for days
- SDS page trial showed that the protein begins to lose integrity quickly
14
Q
UapA Mutagenesis
A
- focus on 4 residues not affecting fold or localisation
- mutated protein non functional to reduce conformational flexibility
- substrate binding to decrease flexibility as well
15
Q
Mutant Screening
A
- 6 mutants generated
- extract proteins in detergent and use SEC for crude detergent
- at higher temperatures the WT protein is lost
- some mutants had much higher stability
- picked one mutant with high protein levels and low aggregates
- mutant has much higher long term stability
16
Q
UapA structure
A
- monomer has dimerization gate domain with core domain for transporting
- xanthine binding in gap of half helices
- substrate translocation done by protein dimer
- protein pulls substrate through domain
