Enzymology P2 Flashcards

Question

What is the usefulness of directed evolution?

Answer 1

Improve protein properties or identify new functions e.g. pH thermal and/or stability pH and/or temperature optima binding properties - altered Km for substrate(s) catalytic properties - enhanced Kcat selection of altered fluorescence properties selection of new binding proteins

Answer 2

Take the gene(s) - don't need to know anything about it, as we are just looking for mutants Introduce diversity - SM, EP-PCR, DNA shuffling, Family DNA shuffling, StEP, RACHITT, ITCHY, ADP, SCHEMA Express many variants in a suitable host (E.coli) Identify suitable variants - chromogenic/fluorogenic substrates, coupled enzyme assays, IVC, Surrogate substrates, growth selections Repeat Until - final improved enzyme is found

Answer 3

We have to be able to physically have the code of the gene for the random variant we selected in order to move onto the next cycle = phenotype-genotype link Phenotype-Genotype link – need to be able to recover the DNA encoding the improved protein Introduce random mutations into the DNA so do not need to decide what mutations to make Error-prone PCR, DNA shuffling Create a library in which each gene copy contains a few mutations Set up a way of screening or selecting improved proteins encoded by the library

Answer 4

In vivo - cell transfection Most common is to use a cell - as it contains nucleic acid we have manipulated and it expresses the protein This restricts the library size to 10^10 due to transfection efficiency In vitro - no transfection step = larger libraries 10^14 Technically demanding The coding sequence must be trapped in a complex with the protein that it encodes

Answer 5

Phage link Yeast or bacterial cell Add a plasmid containing out gene - this cell expresses the protein Or the plasmid can be encoded so the proteins are displayed on cell surface Ribosome display Technically demanding method Stall the complex where the ribosome doesn't fall of the expressed protein - has a peptide linked between the two mRNA The stalled ribosome has made this, forming an artificial covalent link between the peptide and mRNA Also very technically demanding - very highly levels of variants Cis-DNA Oil/water droplets

Answer 6

if we take a water solution containing DNA and a cell extract The DNA can be made into mRNA and proteins - as the water solution will take the DNA to make proteins If we put the water solution into oil we get drops of water within the emulsion Each drop can be modified so it only contains one molecule of water - where each drop contains one DNA molecule and therefore one protein They can work at very high rates - 1000 sec-1

Answer 7

Using a bacteriophage - most common M13 M13 - single strand DNA genome, including any inserted coding region, is packaged in the phage particle It is encoded by proteins forming a coat Proteins can be expressed as translational fusions to coat proteins We can encode our proteins that we want to express into these coats - displayed on the coat major coat protein gp8; 50aa, 3,000 copies – good for peptides minor coat protein gp3; 406aa; 5 copies – good for proteins

Answer 8

Error prone PCR DNA shuffling Family shuffling

Answer 9

``` Reduce PCR fidelity Increased conc. Taq, MgCl2 Increased extension time Altered nucleotide concentration dATP & dGTP 0.2mM dCTP & dTTP 1mM ``` We WANT to make mistakes to force the copying process to make more mutagens Mutazyme II is a blend of Mutazyme I DNA polymerase and a novel Taq DNA polymerase mutant that exhibits increased mis-insertion and misextension frequencies The single amino acid changes are limited to 5 other amino acids + stop codon = limited by codon usage

Answer 10

Error prone PCR is combined with a recombinant method = DNA shuffling From Error prone PCR - produce fragments (using sonication/DNase) Denature them and use a PCR like reaction to anneal and extend through self-priming The sequence is then very shuffled/mixed up Population variation through recombination

Answer 11

DNA shuffling of a FAMILY of sequences should recombine these to generate new variants A high proportion of the products should be functional (folded, stable, display activity) Inactive variants will still exist but should be minority Example - used for biological washing powder methods (serine proteases)

Answer 12

You get what you screen for | In practice we can select or screen

Answer 13

Selection is ideal if a survival phenotype is associated with protein e.g. Growth v no growth in antibiotic resistance We grow the library to see this - if they live they have the enzyme we are looking for But it can be difficult to devise a live/dead selection for many proteins that we might want to evolve It can be used if there is an obvious phenotype in a plate assay = coloured products or a coupled assay of an enzyme with another enzyme system that produces a colour If nothing obvious available we have to screen

Answer 14

Many proteins have no obvious phenotype for bacterial growth or colour formation in a plate based assay Need to screen individual clones for activity; 10^3 -10^5 Need to be careful not to over-mutate - most clones should show activity Enhanced throughput by using robotics but still limited to numbers that can be screened and expense Very inefficient and expensive if most variants are inactive/unfolded so need to ensure relatively low mutation rate often 1-3 aa per gene Has driven the need to generate smaller more focused libraries

Answer 15

Used error prone PCR to mutate the WT gene 2 clones with improved residual activity T34A, S198T, E239G I195V, E239V Higher residual activity by mutating E239 Even after solving the 3D structure this would not have been identified without a random mutagenesis/screen strategy

Answer 16

It is useful to have different colours of the protein We can do directed evolution with GFP All we have to do is look at the fluorescence of the bacterial colonies

Answer 17

CASTing - (Combinatorial Active site Saturation Testing) We take 'groups' of the wild type and screen them We then take the best of the group and then repeat - screening further This isn't perfect as some may need certain combinations that we didn't try in the initial stages

Answer 18

Stereoisomers Different stereo-isomers have different biological effects e.g. Different smell We want to use directed evolution to change the stereochemistry within the active site - this is hard as it is only slightly altering the shape We can use directed evolution as we don't need to understand it fully Enantiomers = mirror images If we have 2 or more 'chiral' centers = diastereoisomers

Answer 19

Through 6 generations of directed evolution we can see an increase in preference to a certain enantiomer - altering an amino acid residue each time (mutation) The first mutation is maintained for the 2nd generation, and each mutation after contains the previous mutation as that type is better - we are trying to improve This was through error-prone PCR They went back through and looked at the combinations of mutations to see if there was a better mutation to a different amino acid - saturation mutagenesis After taking a better mutation - they took this type and did an error PCR to find an even better mutation for a larger 'E' We end up taking a path but there could have been other/better mutations However, the route we go through can't be controlled but it affects what we get at the end - as some mutations wouldn't have been found if it wasn't for the path we took

Answer 20

E (selectivity factor) - if the number is 1 it would favour the 2 enantiomers equally Drawback - not selecting for the real compound we want We are selecting an analogue

Answer 21

Th stereochemical outcome can determine: By which enantiomer was used AND By which reaction mechanism is used Trying to do directed evolution to change these chemical processes - even harder

Answer 22

We can see the different energy levels in two products Therefore we can get a thermodynamic ratio of the products - we can't change this The kinetic ratio - we can alter to change the 'barrier' so more of that product is made

Answer 23

Computational methods E.g. Determine a visual landscape of how the reaction moves along We can see structures that will move along the minimum energy pathway (not up hill)