|Protein expression studies|
Cultivating baculovirus-infected cells for protein expression studies. Any media recommended for insect cells can be used for protein expression studies. For initial studies we seed Sf9 or Sf21 cells as about 90-95% confluent monolayers in 8.8 cm2 Nunclone Delta treated Petri dishes (Nunc Cat. #150318) containing ~2 ml of TC-100 medium supplemented with heat inactivated 10% FBS.
We seed cells from a log phase suspension culture about 1-2 h prior to infection to ensure optimal cell density. Alternatively, cells could be seeded overnight prior to the infection. However, in this case it is more difficult to ensure desirable cell density. Overgrown monolayers with tiny crowded cells are not acceptable as they are dramatically inferior in supporting virus replication and in recombinant protein expression. If overnight monolayears are slightly overgrown, try to dislodge some of the cells by vigorous shaking to obtain about 90% confluent monolayers and change the medium. Less than 75% confluent monolayers are also suboptimal. Obviously, they will yield less protein for analysis as there is less biomass. More importantly, cells which do not have contact with other cells grow less well in Petri dishes, and when infected, they are easily detached from the monolayer.
Infect the monolayers with 300 μl of the initial (P0) virus stock obtained after transfection or with 50-200 μl of a high titer (P1) baculovirus stock. Simply add recombinant baculovirus stock to the monolayer containing 2 ml of the medium and incubate at 28oC for 48-72 h.
For the highest level of recombinant protein expression, harvest baculovirus-infected cells when some of the cells collapse, but at least 90% are still viable (round shaped). This is usually observed after about 60 h post-infection in cell cultures grown on serum-supplemented media, but may vary dependent on the media and on the nature of the recombinant proteins. Lysis occurs faster if a recombinant protein is cytotoxic.
Prolonged incubation (over 70 h) generally is not recommended as it eventually results in death of majority of the cells. Typically, if the maximum protein yield is observed at 60 h post infection, yield at 36 h would be about 10-20%, and at 48 h about 30-60% of the maximum. You may choose shorter incubation times (i.e. 36 h, 48 h etc.) if your protein of interest is largely insoluble or prone to degradation. Alternatively, use ProFold™-C1 vector (Cat #A2) to actively fold cytoplasmic recombinant proteins using human Hsp40 and Hsc70 molecular chaperones encoded by the vector.
In case you grow cells in a serum-supplemented medium but your protein of interest is a secreted protein, you can infect cells in that medium, but change the medium to a synthetic serum-free medium (Lonza’s insect-xpress, BW12-730F or Invitrogen’s Sf-900™ III) 12-24 h post-infection. There is no need to change the medium if cells were infected in serum-free medium. You can actively fold secreted glycoproteins using Calreticulin encoded in ProFold™-ER1 vector if secretion is poor and majority of recombinant protein forms inclusion bodies. Harvest the cells and cell culture medium and investigate for expression of protein of interest by SDS-PAGE, Western blot or ELISA.
For small-scale protein production, especially if the protein expression level is low, we recommend cultivating infected cells in monolayers. In our experiments with various proteins the best recombinant protein expression level calculated per cell or per total cell protein was consistently achieved in monolayers grown on TC-100 medium supplemented with heat inactivated 10% FBS.
In general, switching from monolayers to a suspension culture resulted in a ~2 fold drop in the recombinant protein production per cell. Switching from FBS-supplemented TC-100 to a synthetic serum-free media resulted in a further ~2 fold drop in expression level per cell either in suspension or in the monolayers. Supplementing synthetic serum-free media with various amounts of FBS gave intermediate improvement and generally is not recommended.
Use of synthetic serum-free media (Lonza’s insect-xpress or Invitrogen’s Sf-900™ III) is undoubtedly justified for cost-efficient large scale production of well expressed proteins. Most importantly, serum-free media are indispensable for production of secreted proteins as bovine albumin present in the serum largely complicates analysis of the protein expression and renders recombinant protein purification from the media nearly unfeasible. Be aware that some synthetic media may contain components that interfere with protein purification and may quickly clog an ion exchange column. A clogged column has drastically reduced flow rate and may become stained with dark material adsorbed from the medium. Contact the media manufacture regarding the problem. Better overall performance of synthetic serum-free media is often claimed based on a much higher cell density to which insect Sf9 or Sf21 cells can grow in suspension as compared to growth in the media supplemented with FBS. However, in this case there are more stringent requirements for protein purification as the percent of the protein of interest in the cell extracts is reduced, especially at high densities.
In general, we prefer FBS-supplemented media over synthetic media for initial protein expression studies. High titer recombinant baculovirus stocks can be obtained on synthetic media. However, they quickly loose their infectious activity during the storage. The stability can be improved by adding heat inactivated serum to 2% concentration.
Harvesting cells and cell culture medium for protein expression studies. Remove all medium from an infected monolayer grown in 8.8 cm2 Petri dish into an Eppendorf tube to collect cells that were detached from the monolayer. Collecting the medium is not necessary if the protein of interest is not secreted and the majority of the cells remain attached. Spin the medium in a microfuge at 2,000 r.p.m. for 3 min to pellet the cells which were detached from the monolayer. Remove the supernatant and save it for detecting the protein of interest in case it’s a secreted protein.
Wash the monolayer once with 1 ml PBS, in case the cell culture contained serum. Add the wash to the cells that were pelleted from the cell culture medium, gently resuspend the cells in the wash and spin as above. Completely discard the supernatant. Add 80 μl of TNN buffer (50 mM tris-HCl pH 8.0, 150 mM NaCl, 5 mM EDTA, 0.5% igepal CA-630) to the monolayer. Dislodge attached cells by pipetting the buffer using a Pipetman, transfer the liquid into the Eppendorf tube with the pelleted cells from the wash and the medium and resuspend the pellet. Cell lysis occurs during dislodging and resuspension due to the action of the detergent. Spin the cell lysate at 14,000 r.p.m. for 15 min in a microfuge to separate soluble and insoluble fractions.
Resuspend the pellet (insoluble fraction) in 80 μl of 1x SDS-PAGE gel loading buffer avoiding excessive foaming. Heat for 15 min at 100oC to reduce the viscosity and load 8-15 μl per well for SDS-PAGE. Mix 1 volume of the supernatant (soluble fraction) with 1 volume of 2x loading buffer, heat for 2-3 min at 100oC and load 8-15 μl per well for SDS-PAGE. For analysis of proteins secreted into the serum-free medium, mix 1 volume of the clarified medium (supernatant) with 1 volume of 2x loading buffer, heat 2-3 min at 100oC and load maximum volume per well for SDS-PAGE. Separate proteins according to the gel manufacturer’s instructions and stain with Coomassie blue.
Typically, you’ll see a prominent band with mobility in the gels corresponding to the expected MW of the recombinant protein either in the soluble fraction, or in the pellet or both. Some proteins would appear to have higher MW than calculated since insect cells provide protein modifications characteristic for higher eucaryotes, (most commonly glycosylation, phosphorylation and fatty acid acylation). Usually protein modifications characteristic of a particular protein of interest are known in advance and the extra band(s) of recombinant protein can be easily detected by comparison with a negative control (see below). It is more difficult to detect recombinant proteins in the cell culture medium as the protein concentration is lower. However, only a fraction of the sectreted protein is usually secreted, the rest remains intracellular and usually can be easily detected in soluble or insoluble cell fractions.
In order to identify the recombinant protein band, it is important to run alongside identically processed samples obtained from the cells infected with NC (negative control) baculovirus, which does not express recombinant proteins. To this same end, you can also run extracts of cells infected with any recombinant baculovirus which is expressing a recombinant protein, but of a different size from your protein of interest. We do not use samples obtained from mock-infected cells for this purpose as their protein profiles differ substantially from baculovirus-infected cells.
To confirm identity of the protein of interest, you can cut out the band and submit it for tryptic digestion and Mass-Spectrometry analysis. Alternatively, you can run Western blots or ELISAs if the antibodies to the recombinant protein are available. You can detect several different proteins with the same antibody if the proteins are expressed with the same tag. GST-, His-, GFP-, MBP- FLAG- tag specific antibodies are usually of good quality and are available from several suppliers. Note that proteins present not only in cell extracts and serum-free media, but also in serum-containing media can be reliably detected and quantitated in ELISAs.
|Technology - Maintaining cells - Transfection - Baculovirus stocks - Protein expression|