How long does it take to generate a new baculovirus and test expression?
For the Bac-to-Bac and Baculogold methods, our average time to make the virus, amplify a high-titer stock, and test the kinetics of protein expression is about 3-3.5 weeks. We will notify you by email when we start your time course expression study or production and when you will be able to collect your samples from the Facility.
Is it necessary to plaque purify my virus?
It depends upon the baculovirus transfer/donor vector in which you clone your GOI. Vectors in the BaculoGold™ and Bac-to-Bac™ methods are designed to achieve virtually 100% recombination efficiencies and recombinant protein expression is subsequently evaluated using recombinant virus amplified (without plaque purification) in P2 in insect cells. A single plaque purification of recombinant virus from the initial virus production (P1 virus) is optional before virus is amplified in a second passage (P2) or higher. Recombinant baculovirus derived from all other commercially available baculovirus DNA preparations is produced with 80-90% efficiency and requires plaque purification to remove parental virus.
Does plaque purification of Baculogold or Bac-to-Bac derived viruses enhance the expression of the target protein?
We have looked at a limited number of cases and found that we really do not see significant differences in the expression level (<2-fold) or integrity of a protein expressed from 5 plaque purified viruses derived from a parental polyclonal virus supernatant.
What sort of yield can I expect?
Due to the intrinsic biochemical properties of each individual protein, we do not guarantee recombinant protein solubility, purity, or yield. We will provide details of all expression trials, purification strategies, and provide consultation regarding alternative strategies or approaches to solve your protein expression needs.
How do you control for expression?
The Facility aims to maintain suspension cultures of logarithmically growing Sf9, Sf21, and High Five (T.ni) cells in serum free medium (SFM) with cell viabilities greater than 85%. Deviations from these parameters provide an indication of cell deterioration. Cultures are maintained for no more than ~35 passages before reestablishing new cultures of low passage cells from cryopreserved stocks of the same lot. Infection of insect cells with baculovirus vectors inhibits cell growth and cell viability decreases post-infection. During productions, we monitor total cell numbers and percentage of viable cells as a function of time post-infection.
Why does my protein not express or why are the yields for my protein poor?
Assuming the GOI is properly cloned into the baculovirus transfer, this question is difficult to definitively answer. There are numerous examples where the integrity, stability and biological activity of recombinant proteins vary with time after infection. Therefore, the recommendation of the Facility is that each new baculovirus for a protein be extensively characterized in analytical scale time course expression experiments before proceeding to large-scale productions.
On rare occasions, we have found that expression of a recombinant protein in insect cells can exist in inclusion bodies. We recommend that investigators analyze both soluble and insoluble protein fractions from analytical time course expression studies. For secreted proteins, we recommend that investigators analyze the amount of protein in the supernatant and in cell pellets to assess the extent of secreted protein expressed.
How stable are the baculovirus stocks?
We generally store our high-titer viral stocks at 4°C for up to 6 months. While the stability of some viruses extend beyond 6 months, it has been our experience that the titers of the virus begin to decline. The Facility recommends that a new high-titer virus stock be prepared from existing stocks before the initiation of a new production. In addition, we cryopreserve 1ml aliquots of these stocks at -80°C for long term storage.
Can I use the Facility's equipment for my own cell culture?
In order to avoid cross-contamination, the Facility’s equipment is not be accessible for general use.
Do you purify proteins?
Yes! The Facility provides analytical and preparative scale, one- or two-step purification of recombinant proteins expressed in bacteria, baculovirus infected insect cells, or mammalian cell lines. For more information, please see services.
What is the titer of retroviral vectors you generate?
In our experience the specific titer of a retroviral vector we produce depends upon the total size of the vector, nature of the insert, and quality of the DNA provided. In general, the smaller the total size of the viral vector the better the titers we have been able to generate. We recommend, whenever possible, that newly produced vectors be titrated. We also offer services to concentrate vector preparations. Based on our protocols, we routinely achieve titers for conditioned cell supernatant in the range 1 x 105-1 x 106 TU/ml. For concentrated vector, titers range from 1 x108-1x109 TU/ml.
Do I need to register my intended use of retroviruses with my Institutional Biosafety Committee?
The process of virus packing by the Protein Expression Facility is registered with the Wistar Institute Biosafety Committee (approval # 21102382, exp 3/2014). It is the responsibility of the investigator to register the intended specific use of recombinant DNA technology (i.e. retrovirus use) with Wistar’s Institutional Biosafety Committee (IBC) as required by the most current NIH guidelines.
What percentage of the HIV sequence is retained in the lentivirus vector produced?
Based on DNA sequence comparisons between common LV vectors (e.g. pLKO.1, pLU, pGIPZ) and NCBI accession # K03455, we estimate approximately 10-15%.
How much virus do I need to infect my cells?
The transduction efficiency of tissue culture cell lines is variable. We recommend that investigators determine the transduction efficiency of their specific cells with a titrated virus that expresses a fluorescent protein tracer.
How stable are the retrovirus stocks you prepare?
The stability of each virus should be assessed individually. Based on our experience, conditioned cell supernatants can be stable upto 2 months at 4°C with minimal loss of infectivity.
Does the facility provide service for non-Wistar investigators?
How do you prioritize work requests?
The Facility’s staff strives to accommodate all requests in a timely order from receipt of a service request. The facility has the capacity to handle between 50-60 insect cell cultures (any volume) per week. All work requests are initiated on a first-come-basis, with Wistar Cancer Center members receiving priority, and all other user requests being fulfilled as soon as possible.