I have done BV work for both soluble protein and membrane protein. I would like to recommend you: 1) add some Benzonase to take care of the DNA, this will help you solve the future filter problem; 2) wash with PBS after harvesting cells, "freeze-thraw"procedure may also help to lyse the cell pellets; 3) Increase spinning time, for example you can take the supernatant from first 30min spin and transfer them into new tubes and high-speed centrifuge again for another 30to60min; 4) Increase your lysis buffer volume if your cell pellet is big e.g. if it is more than 5ml cell/liter culture; 5) last point but may not work for your protein, if your single protein is happy in high salt, increase the salt concentration in your lysis buffer, e.g. 500mM NaCl, this may help to remove the aggregates. Hope it helps.

Luigi

I have been purifying proteins from insect cells for about a decade. here is what we do.

For 2.5 L culture, we use about 150 ml of lysis buffer containing 10 % glycerol, 500mM NaCl and Tris 50mM pH 7.9. We then re-suspend the pellet into a fine suspension using a hand held homogenizer. then pass the suspension thru a microfluidizer for 2 cycles. The lysate from the microfluidizer is clarified by centrifugation at 20 k rpm for 1 h. the supernatant is further clarified by 0.2 mic filtration. This supernatant is further used in down streaming application to isolate the protein. We loose only about 5% of sample. You can add benzonase before lysis, if the sample is viscous but microluidization shears all the genomic DNA and you are good to go..

Hope this helps

In a study for in vivo determination of enzyme activities expressed by insect cells we used 1 min sonication at 20% power output (10 secs on, 10 secs off) and it worked well.

Have you verified if the isoelectric point of your protein is close to the pH of the extraction buffer you are using. The protein may be precipitating during the process.

I try to stay way from any detergents in my purifications since we found Triton stays stuck to our protein (according to MS experiments).

We use a lysis buffer containing 5mM TES, 0.2mM EDTA, and 300mM NaCl. And then the cells are broken with a french pressure cell press. We then filter through a 0.2um filter and continue forward with purification with a nickel column.

I would increase the volume of lysis buffer you are using and try the purification again.

I have used a single pass through a french press and 3X30 sec sonication with equal success. We centrifuge all our preps regardless of source at 40,000 rpm for 1.5 hours. We use 10% glycerol in a 25 mM phosphate buffer or Tris buffer at pH 7.4 with 300 to 1 M KCl and 5 to 10 mM EDTA depending on what the next purification step is. I would not increase your volume as that is not really your problem. We do not use detergents except 0.1 % IgePal or other non-ionic detergents. We also don't bother with filtering through a membrane due to the centrifugation that we use. Kent Soe has a good protocol too.

hi

An alternate way to lyse sf9 cells is to resuspend the lysate in the lysis buffer and freeze overnight at -80. Next day, thaw the pellet at room temperature which will burst open the cells. Before thaw, add protease inhibitor and once thawing starts add benzonase to take care of DNA. After complete thaw, a good method is to check for cell lysis using thypan blue staining and if cells are still not lysed, a gentle hand held homogenizer should be enough to lyse the remaining cells. We have used this method to purify full-length kinases from sf9 cells without any problem. This method is detergent-free as well.

Hi Luigi,

If you have worked with E.coli alot then you know that every protein is different. The same goes for Insect cells. I have worked with both with multiple different proteins expressed and purified. One of the more picky proteins we had was a ~70kDa human protein expressed in Sf9. Cells were harvested and frozen in PBS at -80C. Lysis buffer was 50mM PO4 pH 8.0, 300mM NaCl, 10% Glycerol, 10mM Imidazole and 1% Tx-100 with PI. The pellet was thawed on ice in 100 mL lysis and then VERY gently broken up with a 10mL syringe, anything harsher and the protein aggregated, then left sit for 30mins on ice. After centrifugation at 25000rpm IMAC resin was added to the supernatant for a batch purification (unsure what method you are using as you have not mentioned it). The supernatant was always cloudy. When it was clear it never purified. Using IMAC with the cloudy supernatant just meant that it was an effort to purify as the resin regularly blocked up and had to be resuspended frequently...but it worked. I never tried to filter it as I did not want to lose my protein.

So if you are losing protein through the filtration step maybe try a batch purification without filtering. Hope it helps and good luck.

My protein is expressed in the cytosol. I use 1% IgePal detergent in buffer (with imidazole and protease inhibitor) and lately I also freeze the pellet - this seems enough to lyse the cells (insect cells are more sensitive than E Coli to lysis). Lyse 10min on ice, with mixing. I then spin down 15,000 xg for 10min to remove nuclear debris and membranes (this pellet is quite 'sticky'; I find it is a bit looser if I freeze the cells, possible nuclear disruption?). I do not filter the supernatant I just add my Ni-NTA beads to bind my histag protein, bind 2 hours, then wash and elute.

I have noticed this white stuff near the top after spinning a couple times and generally the infected culture had higher opactiy before spinning in this case. I suspected there may be a low-level bacterial or fungal contamination on my hands and this was where the white stuff came from? However I went ahead with protein purification as the cells still looked like they would ordinarily, yield was low (however my infections have given variable yield lately anyway).

I have seen the same white layer on the surface of my cell lysates too-I am extracting cytosolic proteins from whole sandflies using I-Per insect cell extraction reagent (ex Thermo Sci.) This contains a proprietary non ionic sutfactant. I use a Dounce type homogeniser and follow this with centrifugation at 15,000g for 15min at 4C. This is when the white band is evident. I remove the supernatant and spin to disperse this band and follow-on with Mg binding- IMAC using GE Healthcare Hi-Trap columns without back-pressure problems. I wonder if this layer is lipidic material?

I have done BV work for both soluble protein and membrane protein. I would like to recommend you: 1) add some Benzonase to take care of the DNA, this will help you solve the future filter problem; 2) wash with PBS after harvesting cells, "freeze-thraw"procedure may also help to lyse the cell pellets; 3) Increase spinning time, for example you can take the supernatant from first 30min spin and transfer them into new tubes and high-speed centrifuge again for another 30to60min; 4) Increase your lysis buffer volume if your cell pellet is big e.g. if it is more than 5ml cell/liter culture; 5) last point but may not work for your protein, if your single protein is happy in high salt, increase the salt concentration in your lysis buffer, e.g. 500mM NaCl, this may help to remove the aggregates. Hope it helps.

Benzonase will help a lot. Sonication can be harder, 2 to 4 min, ampl can be 30 to 50% according to the situation of your sonicator head (the new changed head, use less power). The lysis buffer, 5 volumes of your cell pellet are generally used. Spin at 20k rpm for longer time, saying 1 hr, also helps. Wash your cell with cold PBS after your harvest the cell also do help, especially when you use nickel-His tag purification strategy. good luck.

Hi all,

Thank you very much for your valuable suggestions. Combining bits and pieces from some of you I think I manage to improve my purification. I have increased the volume of lysis buffer (now I am using a volume of lysis buffer which is about 8 times the volume of the packed cells), increased the centrifuge time to 1 hour and added Benzonase. The combination of those three changes has given me a clear lysate which is not viscous. The increased volume of the lysate makes the first step of purification a bit longer but still doable in half a day. I will probably try some more suggestion later on and will keep you posted.

Thank you

Hi Luigi,

Where does your protein accumulate? For cytoplasmic proteins I do not want to break open the nuclei and release the contents so I use 50 mM sodium phosphate (pH 7.4), 100 mM NaCl, 0.5% NP-40, 5 mM 2-mercaptoethanol and 1X Protease Inhibitor Cocktail at 1-2 mL per 1x10e7 cells. This buffer will not lyse the nuclei. In ice cold buffer NP-40 (and other detergents) can form micelles and that will make the solution look cloudy, but that is not a problem. Triton is a harsher detergent and may lyse the nuclei (hence the need for benzonase).

Imran, you may want to post your question as an independent one, so more people will see it. I have found that NP-40 can be used to efficiently solubilize FIV Envelope, a cytoplasmic membrane protein, when expressed in Sf9 cells. The purity was ~60-70% in one step: incubated cells in ice cold 20 mM Tris pH 7.4 and NP-40 and then spun the cells down, Env was in the supernatant. This was a while ago, but I think I was using ~1-2% NP-40 in the Tris buffer.

Imran,

Typically, 1-2 mL of lysis buffer is used per 1 x 10e7 cells, so for a 25 mL culture you should use 2.5-5 mL of lysis buffer. Are you scaling up in T-flasks or shaker flasks?

Hi Imran,

0.46 mg/mL is okay but how much total protein do you get? And what does the prep look like on SDS/PAGE? Some proteins do not express that well in insect cells. Have you titrated your baculovirus stock and then tested different MOI? (you can do that in 12/24-well plates). MOI can have a big affect on protein expression for some proteins. And have you done a time course for your protein expression? You are harvesting the cells after half have lysed. When an insect cell lyses it releases baculovirus encoded proteases into the media, if your protein is on the cell membrane it could then get degraded. Those baculovirus proteases are released late in infection to lyse the insect cell. A time course starting at 36 hr will tell you how stable your protein is and when is the best time to harvest the cells. I typically harvest Sf9 cells after no more than 25% have lysed, and for some proteins when

Imran,

A high viral titer can be good for one protein and bad for another, that is why you may need to find the titer of your virus and then determine the best MOI. Attached is a paper that shows the effects of different MOI on protein production in Sf9 cells (see Fig. 2).

However, for a long time I made my viral stocks the same way and got good results: infected three T-75 flasks each with ~200-400 uL of a transfection supernatant and let go for 7-9 days or until the cells were >90% lysed. Then took the 45 mL of amplified virus and added to 1 L of cells, let sit for 40 min. with occasional swirling, and then turned on the shaker and harvested the cells around 55-60 hrs.

Hi, if you think filtration is the procedure that you are concerned, why you don't centrifuge at higher speed before filter the lysate? I usually do ultra-centrifugation at 120 000 g for 20 minute, the insoluble material that you observed should be spun down.

Cheers,

Xiaoqin

Dr . Qiuhong Li 

Your protocol is somewhat good. I am plan to follow your protocol to get the higher yield of protein. Please send more details about the protocol and send me the related articles at [email protected]

Hi Luigi,

your sonication protocol is (2 seconds “on” 10% amplitude/60 seconds “off” repeated 15 times), but I have an old instrument from Braun (Labsonic U). It allows sonication with a maximum of 400 W output, so I'd like to know what power level I have to use

Cheers,

Laura

2 cycles in a microfluidizer should be sufficient. please don't add detergents unless you need them as they are impossible to remove especially for structural work

Hi everyone, 

Currently, I am trying to express and purify a peptide (insect peptide) using E. coli method. However, I have not yet made any progress. I am thinking about switching to insect cell line with appropriate plasmid to get this peptide through transfection. I would greatly appreciate if any of you who are expert in this topic willing to share your knowledge with me. In addition, I am humbly asking if you can share your insect cell line and plasmid with me. I am willing to pay any cost that may take part in this process. Thank you so much and I am looking forward to hear back from you guys. 

Luigi,

I would not sonicate the cells "if your protein accumulates in the cytoplasm", sonication will break open the nuclei and release all the DNA making a mess. The Triton X-100 will allow the cytoplasmic contents to drain out. Then do a low speed 2-3 K spin to pellet the nuclei without breaking them open, carefully remove the supernatant, and then do the high speed spin. You can always check the nuclei pellet for your protein, should be 10% or less in it and that is due partly to not being able to get all the supernatant (without contaminating it with nuclei).

Hi Luigi

Did you get this sorted? Is your ouput fine now? If you still need any more trouble shooting tips I am in SW 437 or L4-2581, we specialise in Baculovirus + other cell lines, Protein Purification, characterisation, crystallography, biophysics etc etc

Hi Sara,

Thank you for your answer. Actually that was an old question and I did manage to solve my issue. Since my lysates from insect cells are always very cloudy I started doing the first purification step in a batch mode. So I incubate the lysates with my resin of choice and after a couple of hours of incubation I wash the beads a couple of time in batch. After that I put the beads onto a column and carry on washing for longer. This seems to work totally fine for me. I also managed to publish a paper on the system that I was trying to purify at that time and the full citation is here: The Biophysical Characterisation and SAXS Analysis of Human NLRP1 Uncover a New Level of Complexity of NLR Proteins. Martino L, Holland L, Christodoulou E, Kunzelmann S, Esposito D, Rittinger K. PLoS One. 2016 Oct 11;11(10):e0164662. doi: 10.1371/journal.pone.0164662.

Thank you to you and to all the other helpful comments on this matter.

Luigi