Class 6 E1 Flashcards
What ways can you produce proteins?
- synthesis in a laboratory (chemistry tool)
2. in a cell (biological tool: cloning)
What methods can you use to determine the function of a protein?
protein purification:
- loss of function
- gain of function
- new function.
What methods can you use to characterize the properties of a protein?
Conc. and analyze protein in situ / introduce it into cell / tissue / organism via genetic engineering.
How does solid-phase peptide synthesis (SPPS) work?
- growing peptide coupled to insert resin matrix.
- chemically protected* AA’s added sequentially to growing chain until full length peptide achieved.
- peptide cleaved from resin.
- repeat steps for each AA addition.
The process of SPPS is?
slow, laborious.
What are the caveats of chemical peptide synthesis?
- best for small peptides.
- doesn’t include chaperones / modifying enzymes.
- cellular conditions may be hard to replicate in test tube (type) system.
protein overexpression
use of biological system to produce large quantities of folded protein.
What is an alternative to SPPS and why?
protein overexpression:
- larger size range.
- benefits of other cell components.
What does protein overexpression require?
generating piece of DNA that encodes protein and regulatory elements that ensure protein expression.
What does protein overexpression use?
an organism / cell to express protein after introducing piece of DNA into cell.
expression vectors
DNA that encodes a message for a protein to be expressed and regulatory info needed for that protein’s expression.
expression vectors are made via?
fragments of DNA ligated together in lab to combine elements needed.
What 4 elements do expression vectors contain?
- ori seq
- promoter region
- cDNA coding seq
- polyadenylation seq
ori seq
origin of replication to produce more plasmids.
promoter region
origin of transcription, produce mRNA.
cDNA coding seq
seq to encode protein of interest.
polyadenylation seq
helps ribosomes hang on longer
expression plasmids
tell cells to express.
vectors / plasmids are?
circular DNA, contain promoter, selectable marker, elements that enhance expression / detection.
expression
DNA to RNA to protein.
CMV promoter
viral promoter for gene of interest.
hGH poly(A)
viral poly(A) seq for selectable marker gene.
multiple cloning site
where you can put your gene of interest.
What organisms are used for protein expression?
- Escherichia coli
- saccharomyces cerevisiae
- sf9 cells (army caterpillars)
- HEK cells
site-directed mutagenesis
technique - make small alterations to proteins coding seq via changing DNA.
Site-directed mutagenesis mutations generally affect?
single AA at a time.
What might you want to consider when designing a site-directed mutagenesis?
- desired alteration(s) in AA seq.
2. might not get info on all possibilities.
Why use site-directed mutagenesis?
identify which NTs and AAs are needed to make a functioning protein.
Steps for introducing random mutations to make variants of a protein of interest?
- source DNA inserted into DNA-cloning vector.
- perform PCR rxn using mutagenic primers.
- digest w/ Dpni to cleave original methylated template strands.
- recover mutated product by introducing it into E.coli by transformation.
fusion proteins
generated by in-frame cloning of cDNAs for 2 unrelated proteins / protein domains.
transgenes
used to express fusion proteins.
Transgenes express fusion proteins by?
fluorescent tags (visual) protein of interest fused to easily isolated domain or epitope. enzymes for PCR, lactase, lipases in detergent.
What determines the type of fusion protein in an application?
- where protein is found in cell.
- what domains of protein bind DNA.
- which tissues are promoter active in?
epitope tags
small domains established w/ isolation procedures; make / isolate a lot of your protein.
An epitope tag can serve as?
a label / handle to separate your protein from other cell proteins.
fluorescent fusion proteins
in vivo:
GFP, YFP, CFP, RFP.
