Protein Expression and Libraries Facility

Helpful Information

Baculovirus expression systems
Lentivirus preparation
Vector DB
FAQ

Baculovirus expression systems

DNA Transfection for Baculovirus Expression Vector System 
Spodoptera frugiperda (Sf9) insect cells are cotransfected with the transfer vector (donor or shuttle) plasmid DNA containing the foreign gene to be expressed and BaculoGold™ DNA (PharMingen), Bac-N-Blue™ DNA (Invitrogen), or BacPAK6™ DNA (Clontech).  Alternatively, insect cells are
transfected with a recombinant bacmid DNA constructed by transposition of the donor plasmid DNA in E. coli cells, the so-called Bac-to-Bac™ (Invitrogen-Gibco/Life Technologies) method. The baculovirus DNA is from Autographica californica nuclear polyhedrosis virus (AcNPV). 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. 

Recombinant Baculovirus Stocks
High titer P2 virus stock is produced from the P1 virus (the original virus from cell culture supernatant of co-transfected cells) in Sf9 cells.  P2 virus stock is generally produced in 100 ml of serum-free medium.  A P3 virus (50 ml or 500 ml) is produced by infecting Sf9 cells with a P2 virus at a low multiplicity of infection (MOI=0.1).

Plaque Assay and Purification of Recombinant Baculovirus
Plaque assays are done by infecting sf9 or sf21 insect cells with the P1 virus (or higher passage virus) at a low multiplicity of infection (MOI = 0.1) and overlaying the infected cells with agarose.  Well isolated plaques are scored and virus is subsequently amplified in monolayer cultures of Sf9 cells prior to preparation of larger volume high-titer stocks.

Time-Course Study of Protein Expression 
100 ml suspension cultures of insect cells are infected with a high-titer baculovirus stock at an MOI=1-2.  Cells or conditioned media (for secreted proteins) are harvested at 24, 48, and 72 hours post infection to evaluate the integrity, stability and optimum yield of the product(s) of gene expression. The results can be used to determine optimal conditions for protein production.. Harvested samples are analyzed for expression of the recombinant protein by western blot.  The staff can compare recombinant protein production in at least three different cell lines (sf9, sf21, and High Five) if necessary.

Large-Scale Production of Recombinant Proteins 
High density (1-2 x106 cells/ml) suspension cultures of Sf9, Sf21, or Trichoplusia ni (commonly referred to as High Five) cells are infected at a multiplicity of infection (MOI) equal to 1-2. High Five cells are preferred for production of secreted proteins. We routinely infect cultures (250 ml to 1 L) of insect cells in serum-free medium containing Pluronic F-68 (to prevent cell damage due to shearing) in spinner bottles.
For additional information about insect cell culturing and baculovirus expression systems, please see:
          Guide to baculovirus expression systems and insect cell culturing (PDF)
          Bac-to-Bac Baculovirus expression systems (PDF)
          Baculodirect Baculovirus Expression Systems (PDF)
          Baculovirus Expression Vector Systems (PDF)
          Bac-N-Blue Baculovirus Expression System (PDF)

Lentivirus preparation

HIV-derived lentiviral vectors have been developed as a gene delivery system that can mediate the efficient delivery, integration, and sustained long-term expression of transgenes to dividing and non-dividing cells in vitro and in vivo. Over the years, lentiviral vectors have turned out to represent very powerful tools in basic and translational scientific research. As such, the facility has implemented the infrastructure and technology to produce high-titer lentiviral vectors. In addition, we have pursued their technological improvement for constitutive and conditional expression of cDNAs and shRNAs.

Lentivirus particles are generated by co-expressing the virion packaging elements and the vector genome 
via transient transfection in a producer cell line (e.g. HEK293T). For HIV-1-based vectors, the core and enzymatic components of the virion come from HIV-1, 
while the envelope is derived from a heterologous virus, most often vesicular stomatitis virus (VSV) 
due to the high stability and broad tropism of its G protein. 
By convention, the former elements are referred to as the LV packaging system and the latter as the envelope.

3 components are required to produce an infectious lentivirus vector
• vector, e.g. pLU, pLKO, pGIPZ
• packaging system, e.g. pCMV-dR8.91, psPAX2 (2nd generation)
• envelope plasmid, e.g. pMD2G (VSVg)

Three generations of HIV-based LV packaging systems have been
developed for production of lentivectors by transient transfection.

The first generation LV packaging system encompasses all HIV-1 genes besides the envelope.

The second generation LV packaging system is additionally deleted in all viral auxilliary genes, 
i.e. vpr, vif, vpu and nef. Examples: pCMV-dR8.91, pCMV-dR8.74, psPAX2

The third generation LV packaging system comprises only gag, coding for the virion main structural proteins and pol, responsible for the retrovirus-specific enzymes. A cDNA encoding rev, which encodes a post-transcriptional regulator necessary for efficient gag and pol expression, is provided on a separate plasmid. The third generation packaging system offers maximal biosafety but is more cumbersome, involving the transfection of four different plasmids in the producer cells.
Plasmid DNAs for the 3rd generation packaging system include pMDL g/p RRE and pRSV-Rev.

All lentiviral vectors that contain a wild-type 5'LTR (e.g. pGIPZ) need to be packaged with a 2nd generation packaging system, as wt 5'LTR requires TAT for maximal activation of transcription. The 3rd generation packaging system can only be used with a lentiviral vector with a chimeric 5'LTR e.g. CCL-, RRL-, etc, in which HIV promoter has been replaced with CMV or RSV, thus making them TAT-independent. The lentivectors carrying the chimeric 5'LTR can be packaged into both, 2nd or 3rd generation packaging system.

The production of vector particles by our facility uses a second generation packaging system, which satisfies most applications.

The vector itself is the only genetic material transferred to the target cells. It typically comprises the transgene cassette flanked by cis-acting elements necessary for its encapsidation, reverse transcription and integration. As previously done with oncoretroviral vectors, advantage was taken of the gymnastics of reverse transcription to engineer self-inactivating (SIN) HIV-1-derived vectors, which lose the transcriptional capacity of the viral long terminal repeat (LTR) once transferred to target cells. This minimizes the risk of emergence of replication 
competent recombinants (RCR) and avoids problems linked to promoter interference.

Vector DB (Note this site is under construction.  Links to maps and sequences are incomplete)

Bacterial Expression Plasmids

Baculovirus Expression Vectors

Mammalian Expression Vectors

Lentivirus vectors

FAQ

Following are frequently asked questions the Facility has encountered through the years. Please contact facility staff during hours of operation (weekdays 8:00 am to 6:00 pm EST) at proteinexp@wistar.org or 215-898-3814 with any questions not addressed below.

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.

Will I be able to use the Facility's equipment for my own cell culture?

In order to avoid cross-contamination, the Facility’s equipment will 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 depend 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.
 
Does the facility provide service for non-Wistar investigators?

Yes!

How do you prioritize work requests?

The Facility’s staff strive to accommodate all requests in a timely order from receipt of a service request.  The facility has three 25 cu. ft. upright incubators with stirrers, with the capacity to handle up to 50 spinner flasks of insect cell cultures. 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.