Proteins 2 Flashcards
questions to ask about your protein
disulfides?
exported?
glycosylation?
toxic?
subunits?
detection?
MCS
multiple cloning site
without IPTG, the lac repressor
shuts down the T7 polymerase transcription
without T7, the gene of interest
does not get transcribed and protein does not get made
even without IPTG you can get
leaky expression
there are strains with tighter control of the lac system well suited for
toxic proteins
components of a plasmid
T7 promoter
lac operator
ribosome binding site
his tag
thrombin site
T7 tag
MCS
T7 terminator
types of PCR
simple
overlapping
quickchange
a problem with TAQ polymerase
Taq polymerase has a nontemplate-dependent terminal
transferase activity that adds a single deoxyadenosine (A)
to the 3’-end of the PCR products.
alternative to TAQ
PFU
in overlapping PCR, that is the linker
ser, gly, gly repeats
4 steps in typical cloning
- PCR out GOI using primera with restriction sites
- DIgest ends with restriction enzymes and purify
- digest expression vector with same enzymes and purify
- mix GOI with open vector and add ligase. transform and select via antibiotic resistance
three types of ends a restriction digest can create
5’ protruding ends
3’ protruding ends
blunt ends
3 problems with blunt end cloning
religating vector
orientation
low effiecency
a cute/cheap trick for blunt end cloning
1) Clean the ends of your PCR
with one blunt end enzyme.
2) Open the vector with another
blunt end (here it is SspI)
3) Add SspI to the ligation mix. If
you get empty vector, the
enzyme will cut it open again. If
you get an insert, then the SspI
site is destroyed and stable.
Topoisomerase-based cloning (TOPO cloning) is
a molecular biology technique in which DNA fragments
are cloned into specific vectors without the requirement
for DNA ligases.
The biological role of topoisomerase is to
cleave and rejoin supercoiled DNA ends to facilitate
replication.
The major disadvantage of TOPO® cloning is that
very few plasmid backbones are available TOPO® ready, and it
is not feasible to create a TOPO® vector yourself.
Additionally, the efficiency can vary depending on the polymerase used, and the single A overhangs degrade over time, further reducing ligation efficiency. TOPO® ready Gateway® Entry plasmids are also available,
allowing for rapid cloning of PCR products into donor plasmids without the need for restriction enzyme cloning.
blue/white selection
white cells have foreign gene inserted into plasmid.
blue cells have plasmid but not the foreign gene
heat shock transformation
uses a calcium rich environment provided by calcium chloride to counteract the electrostatic repulsion between the plasmid DNA and bacterial cellular membrane. A sudden increase in
temperature creates pores in the
plasma membrane of the bacteria
and allows for plasmid DNA to enter the bacterial cell
Electroporation, or
electropermeabilization,
is where an electrical field is
applied to cells in order to
increase the permeability of
the cell membrane, allowing
chemicals, drugs, or DNA to
be introduced into the cell.
quickchange pcr only works if
your plasmid is small
quickchange pcr uses what to digest the template
DpnI
The #1 Commandment of cloning and expression:
SEQUENCE
A possible problem in expression can be
that codons commonly used in
animals are often rare in bacteria.
inclusion bodies
how to fix rare codon problem
rosetta strians or additional vectors
advantages of periplasmic expression over cytoplasmic production
1: An authentic N terminus can be obtained after removal of the signal sequence by leader
peptidases.
2: The periplasm is conducive to disulfide bond formation because of the presence of the Dsb (thioredoxin family) machinery.
3: There are fewer proteases in the periplasm compared to the cytoplasm and many have specific
substrates.
4: Because the periplasm contains fewer proteins and because its content can be selectively
released by osmotic shock or other strategies purification of the target protein is facilitated.
highlights of NEB SHuffle
Engineered E. coli K12 to promote disulfide bond formation in the cytoplasm
*Constitutively expresses a chromosomal copy of the disulfide bond isomerase DsbC that promotes the correction of mis-oxidized
proteins into their correct form
*The cytoplasmic DsbC is also a chaperone that can assist in the folding of proteins that do not require disulfide bonds
*Expresses a chromosomal copy of T7 RNAP
*Tight control of expression by lacI q allows potentially toxic genes to be cloned
*Resistance to phage T1 (fhuA2), Nit, Str, Spec
Sometimes, putting a tag on
the protein can help it to
fold and/or prevent aggregation -
perhaps by covering up a
greasy spot on the protein.
Other ways to improve folding/prevent inclusion bodies
decrease the temperature
decrease the induction
Can add 1-3% ethanol or 10-30% glycerol. Both slow down growth and glycerol may help keep the protein soluble.
Ecoli pros
1.Easy to transform with plasmids
2.Cheap and fast growing
3.Strong and inexpensive expression systems (e.g. IPTG)
4.Wide array of tags and fusion protein options
5.Great for Se-Met and isotope labeling
6.Possible periplasm export for better folding (e.g. pelB, OmpA, DsbA, TolB and MalE)
7.Can co-express with GroELS to improve folding
8.Expression can be controlled with temperature
9.There are expression vectors (e.g. Rosetta) for rare tRNAs not predominant in coli
Ecoli cons
1.Can form inclusion bodies
2.Often issues with proteins with disulfides
3.No glycosylation or other eukaryotic post translational
modification.
ampR
ampicillin resistance for bacterial selection
AOX1 promotor
aldehyde oxidase 1 (for yeast methanol induction)
alpha mating factor
secretion signmal for export in yeast
nourseothricin
antibiotic resistance marker for yeast
TEF promotor
translation effector factor (constitutive)
yeast expression can have what kind of colored selection
red/white
yeast pros
1.Easy to transform with plasmids
2.Cheap to grow and fast growth rates
3.Strong and inexpensive expression systems (e.g. MetOH)
4.Wide array of tags and fusion protein options
5.Not bad (not E coli levels) Se-Met and isotope labeling
6.A number of secretion signals with leader processing
7.More like eukaryotic folding
8.Protease deficient strains
9.Glycosylation (but not sialic acid)
yeast cons
1) Slower to grow and transform than bacteria
2) Harder to crack open the cells than bacteria
3) More expensive to label (i.e. NMR) than bacteria
4) Not quite as many vectors and tools
baculovirus expression
Ligate gene into ‘donor’ or ‘shuttle’ vector, express vector in bacteria and purify. Transform shuttle vector into special DH10Bac E coli cells that contains the bacmid (a big chunk of the baculovirus genome) and a helper phage to help with the transposition.
With the flanking regions of the shuttle vector, you can get recombination in the bacmid. With the Lac gene, you can select blue/white colonies. Grow them up and purify the bacmid. Add bacmid and helper virus to make full infectious virus in the insect cells. Select, grow, express protein
insect cells and baculovirus pros
1.Better post-translational modification system
2.Better folding of mammalian proteins.
insect cells and baculovirus cons
1.Very expensive (media, plasticware, etc).
2.~2 weeks for expression
lentivirus expression
Lentiviruses (e.g. HIV) can
integrate genes into the genome.
You make your shuttle plasmid
and then co-infect into the
animal cell line. With the help of
the other plasmids, virus is made
and package your gene. Isolate
virus and infect into new cell line.
animal cells pros
1.Both transient and stable cell line production
2.Very good at making secreted proteins
3.Lentivirus expression systems available for both transient
and stable expression
4.Truly native expression.
animal cells cons
1.Very time consuming
2.Not cheap
two ways to make transgenic plants
agrobacterium or gene gun
plants pros
1.Can make grams of protein
2.No cell culture - seeds and dirt
3.There are some targeted export signals
known
plants cons
1.Very time consuming
2.Transformation not straightforward
3.If plastid expression, often the same as E. coli with regard to folding problems
ways to store proteins
1) Precipitate with ammonium sulfate (e.g. 50-60% saturation). Can be stored at 4°C.
2) 10-50% glycerol and frozen at -20°C or -80°C
3) Lyophilized (if you are REALLY lucky)
4) Short term storage with antimicrobials (e.g. azide) and protease inhibitors (e.g. EDTA and protease inhibitor cocktails).
5) Need to know its ‘happy buffer’ (e.g. pH, phosphate versus Tris, and salt concentrations).
whats the easiest expression system to use
ecoli
what expression system might you use for glycosylated proteins
anything but ecoli
what are the issues with disulfides and how would you fix them
wrong disulfide bonds leads to misfolding
periplasm expression
change cell line
change expression system
chaperones
how might you deal with eukaryotic proteins that are secreted by the cell
flagellar export system
mating export system
golgi
etc
what happens when ecoli expresses protein too high
inclusion bodies
