In both eukaryotic and prokaryotic cells, all proteins are synthesized in cytoplasm.
Proteins that are destined to enter into the secretory pathway are usually endowed with an N-terminal signal peptides (SPs, known also as N terminal leader sequences): the signal peptide those are short peptides and usually have a length of 16–30 amino acids.
After directing proteins to their specific locations, SPs are removed by signal peptidases
The presence or absence of the SPs allow to
direct the expression of the protein in different cellular compartments:
- E.coli,
a protein with-out SPs will be directed in the cytoplasm, while the
addiction of a SPs (as pelB, OmpA signal peptides) of the signal will direct the protein into the periplasm;
-In Gram positive bacteria (as baccilus) and
mammalian cells (as HEK293, CHO) the addiction of N terminal SPs direct the
protein in the culture surnatant.
Since the accumulation of recombinant proteins
in the cytoplasm may lead to the formation of inclusion bodies or protein degradation
via proteases and the recombinant protein folding may also affected from the
reducing conditions of the cell compartment (eg E.coli cytoplasm is strongly
reducing and not compatible with S-S bond formation), the selection of the
right Signal peptides play a decisive role in the industrial production of
recombinant proteins.
It has been shown that using different homologous or heterologous signal
peptides can affect the yields of recombinant proteins. Selecting a proper
signal peptide to increase the secretion efficiency becomes a common
methodology to optimize the production of recombinant protein
The availability of a simple cloning method to readily add, replace and modify a signal peptide sequences in an expression clone is therefore an essential tool to screen for the best
protein/antibody productivity.
As already mentioned in the ProteoCool n°1: Cloning methods overview; The PIPE cloning is a
nice method to manipulate expression vectors and perform mutagenesis,
insertion and/or deletion or some vector regions.
With a single vector PCR is possible to insert dna fragments up to 80-100bp in any vector region
and therefore it can be applied also to the insertion of replacement of signal
peptides in the vector of interest.
PIPE cloning steps:
1) Vector amplification by PCR
(V-PCR)
2) PCR digestion with dpnI (to remove template vector background)
3) Trasfrom the vector into the MACH1 E.coli cells
4) Plasmid extraction from at least 4 colonies and DNA sequencing
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Tips to perform the Vector PCR:
PCR reaction:
My preferred DNA polymerases: Kapa Hifi (Roche) or Clone amp (Takara)
Theoretically you can use all high fidelity polimerases that do not add poly AA
DNA template --> less than 0.1ng/reaction
(higher template amount may
result in background colonies with the original template)
PCR volume --> 25ul/reaction are more than enough considering that
for PIPE reaction 1-2ul are normally enough and PCR purification is not
required.
PCR cycle:
Elongation time --> >1minute/kb also if the datasheet of the Taq
suggest shorter extension time to exploit the 3-' --> 5' exonuclease
activity that improve the formation of incomplete extension that are require
for PIPE cloning.
Do not insert the final elongation time step.
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V-PCR trasfromation in E.coli cells (PIPE reaction)
PIPE cloning do not require PCR purification.
1) Mix 2 ul of the V-PCR in 20ul
of chemically competent MACH1 cells (thermo cod. )and incubate the mixture in
ice 30 minutes;
N.B: If the V-PCR band intensity is very high (as in the reported picture) yon can try also to dilute the plasmid 5
times and perform a second trasfromation with 2ul of the diluted plasmid in
20ul of cells because too much DNA can reduce the trasformation efficiency)
2) Incubate the
cells 1' at 42°C
3) Transfer the
cell in ice
4) Add 250ul of
SOC or LB sterile media (with out antibiotic)
5) incubate the cells
at 37°C - 180/600rpm (in a thermomixer or incubator shaker)
6) Plate all the
cells in LB-agar plates containing the proper selection antibiotic
(eg 100mg/l
ampicillin for pet21 or pcdna 3.4,
50mg/l of kanamicin for pet24 clones)
7) incubate the plates
O/N at 37°C
Primers design
Annealing regions
à 18-26bp with an annealing
temperature salt adjusted (calculated with Oligocalculator) of 58-62°C if is possible)
Flanking regions –>
length up to 60bp. Overlapping regions have to be between 15 and 20bp
Is not mandatory to add flanking regions in both forward and reverse primer to create the 16bp overlapping. It can be done also adding the flanking region in just 1 of the 2 primers. I choose one or the other solution on the basis of the length and Tm of the relative annealing regions.
---------------------------------------------------------------------------------------------------------------------------------------------------- A great thanks to
an amazing supervisor who introduced me to the PIPE cloning
i was very lucky to shave with him many years in the Novartis Reseach centre in Siena
References:
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