Lecture 5: Protein Engineering Flashcards
Protein engineering applications
- Pharmaceuticals
- Agriculture
- Research
- Bioenergy
- White biotech
Rational design of proteins
- Based on protein knowledge
- Structure
- Mechanisms
- Dynamics
- Natural variation
- Analogous to mechanical engineering
Random mutagenesis or directed evolutionary processes
- Library of potential mutations n screen them
- Important to test n isolate for targeted properties
- Once mutant of interest is isolated, characterize what it can do
PEGylation advantages in protein engineering
- Flexible hydrophilic coat → solubility
- Reduced accessibility → protease resistance n non-antigenicity
- Increased size → serum half life
Fluorophores
Fluorescent labelling → tracking location or dynamics
Uricase
- Important in the treatment of gout
- Poor half life in the body
- Attach PEG to it to combat this issue
- Effectively reduce the antigenicity (recognition by our immune system) -> lives longer in the blood stream
- Poor half life in the body
- Take uricase n attach by surface AA to produce PEG-uricase which will degrade uric acid thus getting rid of gout
Rational design of protein by site directed mutagenesis
- Couple of DNA oligonucleotides which will bind to DNA target of interest
- Specify mutant residues required for mutation
- PCR experiment where production of target is increased
- Enzyme to degrade the original template → end up w only required mutant DNA to transform into cells
Structure of DapF
- Changes an L form to a D form
- Heart is a cystine residue
- Cystines evolved in a disulfide bond
- Active site cystine 73 is disulfide bonded to cystine 217 as a result of oxidation upon purification, preventing any enzymological study (dead enzyme)
Rational design of protein by creating fusion proteins
- Creation
- Remove stop codon of first genes
- Ligate genes together in frame
- Include linker codons
Aims of creating fusion proteins
- Combine the properties of the components
- Addition of antibody Fc fragment to proteins increases serum half life
- Co-localize the components
- Set of enzymes that work in a reaction pathway
Considerations for creating fusion proteins
- Linker length n flexibility
- Distance b/w protein components
- Protease resilience
- Ability for domains to fold
What is the functional relationship between PBP2 and RodA in bacterial cells?
- PBP2 and RodA are two membrane proteins in bacteria that work together as part of the biosynthesis of the bacterial cell wall.
- They form a complex, and it was predicted that the C terminus of PBP2 is located in the cytoplasm, likely adjacent to the N terminus of RodA.
How was the spatial relationship between the C terminus of PBP2 and the N terminus of RodA investigated?
- A fusion protein with a small peptide linkage between the C terminus of PBP2 and the N terminus of RodA was created.
- The expressed protein was then used to analyze the spatial arrangement, providing insights into their proximity and interaction.
What technique was employed to solve the cyro EM structure of the PBP2-RodA chimera, and what key features were revealed?
- The membrane scaffold nanodisc was used to solve the cyro EM structure of the PBP2-RodA chimera.
- The structure revealed single transmembrane helices of RodA and identified a red helix that activates RodA, representing the active enzyme in the complex.
Directed evolution in the context of proteins
Using techniques that mimic natural evolution to accelerate the generation of protein variants with potentially new properties of interest
How can we generate variation?
- Introduce a mutation once every 100bp -> creates a library
- End up w lots of point mutations in the library which you then screen
- Shuffle bits of different genes together
- PCR n genetic based strategy
- Selectively take out lumps of DNA to give you variation
Aim of random point mutations
Introduce a random number of mutations into a target gene w the intention of selecting for a new property of that protein (e.g. solubility of heat stability)
Random point mutation methodology
- Thermostable DNA polymerase during PCR, which introduces incorrect nucleotides under specific conditions.
- The number of mutations (usually 1-10 nucleotides/kbp) can be manipulated, generating a library of mutants that is then screened to identify variants with the desired property.
- This approach contrasts with the targeted and specific nature of site-directed mutagenesis (SDM)
How do you screen the mutant library?
- Depends on targeted property
- New functional enzyme/immunological/functional assay → high throughput assay
- Biophysical property: heat stability, pH stability
How can the solubility of a mutant protein be screened effectively, especially when looking for enhanced solubility?
- Screen for enhanced solubility is to fuse the mutant protein to another protein that is intrinsically insoluble.
- This fusion partner should be easy to identify, such as having a distinct color or fluorescence (e.g. GFP)
DNA shuffling
- Gene first cut into pieces using exonuclease then regenerated using a DNA polymerase
- Polymerase introduces mutations similar to error-prone PCR
- DNA pieces get mixed → mutations from separate copies can be combined
- RESULT: library of mutants subject to similar screening methodologies
How does DNA shuffling mimic natural evolution, and what distinguishes it from site-directed mutagenesis (SDM) and random mutagenesis (RM)?
- DNA shuffling mimics natural evolution by recombining genetic material, similar to the process observed in some organisms.
- Unlike artificial methods such as SDM and RM, DNA shuffling involves the creation of genetic diversity through recombination, reflecting a more natural approach to evolving protein
Provide an example of DNA shuffling application and its goal in protein evolution.
- DNA shuffling has been employed in evolving a class C cephalosporinase, a ß-lactam degrading enzyme, derived from four different bacterial species.
- The objective was to produce an enzyme capable of degrading a chemically related ß-lactam antibiotic, moxalactam, showcasing the versatility of DNA shuffling in evolving enzymes for specific functions.
Describe the two strategies adopted in the evolution of the cephalosporinase enzyme and their outcomes.
- Strategy I: individual genes from four different species underwent shuffling, introducing point mutations. The best-selected variant showed an 8-fold increase in resistance to the new drug.
- Strategy II: gene fragments from all four species were mixed in the DNA shuffling experiment, resulting in “new” proteins that exhibited between 270-540 fold more resistance to the new drug. The most effective enzyme in this strategy had eight discrete DNA segments from three of the four genes and 33 additional point mutations.