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Glycosylation patterns in insect
vs. yeast cells
What are the glycosylation patterns and differences between
Pichia, mammalian, and insect cells (Sf9, Sf21, HighFive)?
Pichia glycosylation consists mostly of short chain Man(10)
GlcNAc residues. As compared to other yeast like S. cerevisiae, which tend
to hyperglycosylate, Pichia glycosylation is closer to the typical mammalian
high-mannose glycosylation pattern. O-linked oligosaccharides are present
but are not major components of the total soluble gylcoprotein of Pichia.
As for baculovirus, infection with the virus alters normal glycosylation
and oligosaccharide processing characteristics of insect cells. The nature
of N-linked glycosylation is dependent on the protein expressed, host cell
line used and the length of time of infection of cells with baculovirus.
N-linked glycosylation is generally of the high-mannose type. O-linked
glycosylation is, although not identical, similar to mammlian cells, depending
on localization and type of protein.
References on glycosylation in Pichia:
What's is the best protocol to
lyse insect cells?
What's is the best protocol to lyse insect cells? Can
the French Press be too agressive for the preparation of the membrane-bound
proteins?
The French Press is absolutely unnecessary and not recommended
for lysing cultured cells. Cell extracts may be prepared by a variety of
procedures and in a buffer of choice.
First try a simple freeze-thaw in a buffer lacking detergent
(e.g., PBS). Usually once is enough to obtain cell disruption, but check
by examination in a microscope and repeat as necessary, followed by centrifugation
to remove particulate products.
For proteins that remain in the particulate phase, subsequently
extract the pellet with a buffer that contains a mild detergent. DNA is
usually not a problem when processing cells late in the baculovirus infection
cycle, but if the sample is viscous, briefly vortex to shear the DNA, or
sonicate to reduce viscosity. Never sonicate proteins that are in detergent
as this will cause foaming of the sample. This will usuually kill the protein.
[Be aware that sonication may be deleterious to some proteins and structures.
Avoid heating by keeping the samples on ice during sonication.]
The use of detergent will liberate some membrane proteins,
which may prove difficult to remove at subsequent steps, depending on the
protein. A common lysis buffer is 20 mM Tris-HCl,pH 7.5, 150 mM NaCl, 1%
Triton X-100 (1X Bind/Wash Buffer), containing protease inhibitors. Use
a ratio of 1 ml lysis buffer per 1 x10**8 cells (or less if desired). Incubate
the mixture on ice for 45 minutes. After centrifugation, determine the
distribution of the protein. Alternatively, to avoid nuclear lysis, substitute
1% NP40 for the Triton X-100. Centrifuge at low
speed to pellet nuclei and insoluble proteins.
If the protein remains in the particulate phase, try addition of 1% deoxycholate to the detergent mixture and repeat the extraction. Also try high salt extraction (various salts and concentrations up to 2 M, depending on the salt), and determine the effects of different buffers at different pH values on solubilizing the protein.
For proteins that may be bound to nucleic acids, try the appropriate nuclease treatment, followed by salt extraction. For nuclear proteins, try using 1% NP40 to solubilize plasma membrane and cytosolic proteins.
If all else fails and the protein remains insoluble, it may be necessary to resort to a harsher denaturation protocol (e.g., 6 M urea)after all the soluble products have been removed. After incubation in 6 M urea, dilute to 2 M urea and centrifuge or filter.
Choose another Category of Insect FAQs
How long can S2 cells be induced
with CuSO4 before the CuSO4 has a detrimental effect on the cells?
S2 cells have been maintained in the presence of 200
uM CuSO4 for 4 months without any apparent detrimental effect to growth.
These cells have maintained their fully induced levels of expression (Johansen
et al, Genes and Development 3:882-889).
Calcium phosphate transfection
of S2 cells: important considerations
Is it normal to still be able to see precipitate on Drosophila
S2 cells after a calcium phosphate transfection, even post-washing? What
are the important considerations for transfection?
It is not uncommon to see precipitate for several passages,
however it is gradually diluted away. Occasionally, the Calcium Phosphate
mixture has been left on the cells up to five days yet protein expression
is unaffected. Cell growth may not be optimal and the effects are cell-type-specific
but it is unlikely that cell death will result from prolonged exposure
to the diluted precipitate. Cell death could be due to splitting/diluting
the cells to far back or concentrating the cells. S2 cells should be seeded
at 1 million cells/ml, at 6-16 hours before transfection. 2-4 million cells/ml
is an ideal density for transfection. However, even 4-6 million cells/ml
final cell density would work for transfection. It is important to transfect
cells using media with serum in it. In order to determine whether the cells
are actually dead, a trypan blue exclusion is recommended.
What are the optimal pH and osmolality
values for culturing insect cells?
The pH of a growth medium affects both cellular proliferation
and viral or recombinant protein production. In most applications a pH
range of 6.0 to 6.4 works well for most lepidopteran cell lines. The optimal
osmolality of medium for use with lepidopteran cell lines is 345 to 380
mOsm/kg. To maintain reliable and consistent cellular growth patterns and
minimize technical problems, maintain pH and osmolality within the ranges
listed here.
Seeding densities of Sf9, High
Five, or Sf21 cells
How many Sf9, High Five, or Sf21 cells are used to "seed"
a flask at 50% confluence?
Sf9 cells: Typical seeding densities for 50% confluence
are as follows:
5 x 105 cells/well 12-well plate
1 x 106 cells/well 6-well plate
2 x 106 cells/flask T-25 cm2
6 x 106 cells/flask T-75 cm2
2 x 106 cells/plate 60 mm plate
5 x 106 cells/plate 100 mm plate
1.8-2.2 x 106 cells/mL all spinner flasks
Sf21 cells: These cells are slightly larger in size than
the Sf9 cells so the quantity of cells used to "seed" a flask may be reduced
but this is not essential. The only exception is when you do a plaque assay
with Sf21 cells. Then you should use AT MOST 4.8 x 106 cells per 100 mm
plate as noted below. Otherwise, your plaques will be "overgrown" on the
plates and screening the plaques can become difficult.
Sf121 cells (50% confluence):
4.8 x 105 cells/well 12-well plate
9.6 x 105 cells/well 6-well plate
1.9 x 106 cells/flask T-25 cm2
5.7 x 106 cells/flask T-75 cm2
1.9 x 106 cells/plate 60 mm plate
4.8 x 106 cells/plate 100 mm plate
1.8-2.26 x106 cells/mL all spinner flasks
High Five cells: These cells are often loosely
adherent. They are confluent when they cover about 80% of a given surface
area. It is recommended to seed flasks / plates at 30 - 50% confluence
instead of 50%. This will not be too sparse for the cells. Here are the
adjusted numbers for 30% confluency:
High Five cells (30% confluence):
1.5 x 105cells/well 12-well plate
3 x 105 cells/well 6-well plate
6 x 105 cells/flask T-25 cm2
1.8 x 106 cells/flask T-75 cm2
6 x 105 cells/plate 60 mm plate
1.5 x 106 cells/plate 100 mm plate
1.8-2.26 x 106 cells/mL all spinner flasks
Antibiotics, antifungals used
in insect cell culture of Sf9, Sf21, and High Five
What antimicrobials may be used in insect cell culture
of Sf9, Sf21, and High Five cells? What concentration should be used?
Gentamycin: 10 µg/mL Inhibits bacterial protein
synthesis
Penicillin-Streptomycin:
100-200 U/mL Penicillin Inhibits bacterial cell wall
synthesis
100 µg/mL Streptomycin Inhibits bacterial protein
synthesis
Amphotericin B: Fungizone 0.25 µg/mL Binds sterols
and interferes with membrane permeability
These concentrations are assuming the use of serum-containing media. For serum-free formulations, decrease the antibiotic concentrations by at least half.
How are Sf9, Sf21, and High Five
cells cultured in
selenomethionine containing medium?
Selenomethionine is toxic to Sf9, Sf21, and High Five
cells. To circumvent the toxicity problem and substitute methionine residues
by selenomethionine, the following procedure was performed:
Culture and maintain Sf9 insect cells in complete TNM-FH
medium with 10% FBS
Co-infect Sf9 cells with recombinant virus(s) at an MOI=5-10
pfu/cell. Note: MOI in literature has ranged from 1-10.
36 hours post infection, the Sf9 cells are washed and
incubated with methionine minus Grace?s Insect Medium for 4 hours.
Grace's Insect Medium minus methionine is supplemented
with 100 mg/L L-selenomethionine and 5% FBS for another 36 hours. 84% of
methionine residues were substituted by selenomethionine NOTE: The incubation
times in each of the media will differ with the post-infection harvest
time of the protein being expressed.
This information was gathered from the following paper:
Chen WY, et al. Selenomethionyl analog of recombinant human choriogonadotropin.
J Biol Chem. 1991 May 25;266(15):9355-8. PMID: 2033036; UI: 91236694
How many passages can you go with
Sf9 cells before you should go back to a frozen stock? Does the infectability
of the cells drop off as the cells are passaged more?
Typically you should return to a frozen stock when cells
have undergone 30 passages. Whenever the cells are counted, you should
check for cell viability. If the cells are maintaining a viability above
~95% and are doubling ever 30 hours or so, then they are still OK to use.
If the viability and the doubling time drops, then their infectibility
will not be efficient.
Post-translational modifications
observed in Sf9 cells
It has been reported that methylation of proteins DOES
occur in Sf9 cells. Other modifications that occur are: Glycosylation (N-
and O-linked), cleavage, formation of disulfide bonds, myristylation, palmitation,
acylation, and phosphorylation. It has also been reported that proteins
do all of the above plus: alpha amidation, oligomerization and assembly,
N-terminal blocking, cleavage of signal peptides, internal proteolysis
(specific) occurs.
Co-expression of multiple recombinant
proteins using DES
Have two recombinant proteins been expressed simultaneously
in the DES system?
One example is the expression of human IgG antibodies
(Kirkpatrick et al., 1995 JBC 270:19800-19805). This was achieved by co-transfecting
two separate plasmids encoding heavy and light chain in a 10:10:1 ratio
with pCoHygro. This resulted in the secretion of fully folded, functional
antibodies.
Co-infection of insect cells
with multiple recombinant baculovirus
Can more than one baculovirus particle infect a single
cell? Is it possible to co-infect Baculovirus insect cells with two or
more different recombinant viruses in order to co-express a multi-subunit
protein?
Coinfection does occur. It has been observed where there
is both recombinant and wild type infection. For example, in the Bac-N-Blue
system, a blue plaque with a white center would indicate coinfection. Below
are some references for expression of multimers via infection with multiple
baculovirus stocks. Several five subunit proteins such as human replication
factor C have been expressed. Invitrogen recommends that a separate High
Titer Stock (HTS) of each subunit be produced in order to optimally express
the multi-subunit protein. This way, the amount of each subunit expressed
can be controlled by varying the Multiplicity of Infection (MOI) of each
subunit HTS.
Heterodimer:
Serum-free medium: The following protocol was take from : Weiss SA, et al. Insect cell culture in serum-free media. Methods Mol Biol. 1995;39:79-95. Review. No abstract available. PMID: 7620562; UI: 95345950.<br>It is recommended that the serum-free media (SFM) formulation contain 0.05 - 0.1% Pluronic Polyol F-58 or a polyol equivalent in performance that prevents shearing. Orbital shaker apparatus must have a capacity for 50-500 mL Erlenmeyer flasks and a shaking speed of up to 140 rpm. The standard flask employed is the 250 mL disposable sterile Erlenmeyer for a 100 mL volume. The orbital shaker flask assembly should be maintained in a 28 +/- 0.5°C non-humidified, ambient air-regulated environment. Oxygenation / aeration is accomplished by loosening the cap approximately 1/4 turn (within the intermediate closure position). In this condition, there is no oxygen limitation, and therefore, they proliferate to maximum rates. Inoculate a 250 mL Erlenmeyer flask with 100 mL of complete SFM containing 3 x 10e5 viable cells/mL. Set the orbital shaker at 135 rpm for cultures maintained and adapted to suspension culture in SFM. Incubate the culture in SFM until it reaches 1 - 3 x 10e6 viable cells/mL. Then split the shake flask cultures to approximately 3 x 10e5 viable cells/mL. For consistent optimal cell growth, the culture should be in midlog phase of growth when subcultured. Once every 3 weeks, cell suspension from shaker cultures may be gently centrifuged at 100 x g for 5 minutes, and the cell pellet resuspended in fresh SFM. This reduces accumulation of cell debris and toxic waste byproducts.
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