VE-BEVS Transfer Vector Resource Page

For Technical Support Please Contact Us
Email: info@paratechs.com or Call (+1) 859-317-9213

• pAcVE1 Instructions [PDF]
• pAcVE.02 Instructions [PDF]
• pAcVE.03 Instructions [PDF]

• VE-BEVS Frequently Asked Questions [PDF]
• VE-BEVS Publications
• VE-BEVS Presentations
• VE-BEVS Licensing

pAcVE1 Instructions

Instructions for Product No.: 20010 Qty: 10µg

Description

ParaTechs’ pAcVE1 baculovirus transfer vector is designed for high level expression of foreign genes under the control of the Autographa californica nuclear polyhedrosis virus (AcMNPV) polyhedrin promoter. Increased recombinant protein production over conventional baculovirus transfer vectors is achieved by allowing co-expression of foreign genes with a vankyrin expression cassette (Fath-Goodin et al., 2006). pAcVE1 is derived from the pUC57 vector. Foreign genes may be cloned into the multiple cloning site utilizing the following restriction sites: AvrII; SbfI; XhoI; BglII; EagI; NotI; NheI. pAcVE1 baculovirus transfer vector is a polyhedrin locus-based transfer vector that is compatible with any baculovirus system that utilizes homologous recombination in insect cells, including flashBac (Oxford Expression Technologies) and Bac-N-Blue (Invitrogen).

Contents

The plasmid DNA was prepared on a silicon bead matrix and dissolved in TE buffer (10mM Tris-HCl, pH7.5; 1 mM EDTA). pAcVE1 baculovirus transfer vector is provided at 10 µg in 20 µl.

Storage

Transfer vector should be placed at -200 C for long- term storage.

Handling

For expression of recombinant protein under the polyhedrin promoter, the gene of interest should be ligated into an appropriate restriction site in the multiple cloning site of the pAcVE1 vector (see below).  Transform the recombinant pAcVE1 transfer vector in E. coli cells (Top10 or any other suitable strain), propagate the cells under ampicillin selection and purify the recombinant plasmid using standard plasmid purification protocols.  For construction of recombinant AcMNPV virus perform a co-transfection of the purified recombinant pAcVE1 vector with linearized baculovirus DNA suitable for homologous recombination in insect cells.

pAcVE1 Vector Map and Multiple Cloning Site

Unique Restriction Sites

AgeI   AlwNI   ApaI   AscI   AvrII   BglII   BsaAI   Bsp120I   BssHII   BstAPI   BstXI   ClaI   DraII   EagI   EcoNI   EcoRI   FspAI   MscI   NaeI   NdeI   Ngo   MIV   NheI   NotI   NruI   PacI   PfoI   SbfI   SgrAI   SmaI   SnaBI   SpeI   StyI   SwaI   XhoI   XmaI

Absent Restriction Sites

AflII   BclI   BlpI   BsiWI   BstEII   BstZ17I   Bsu36I   DraIII   FseI   MboI   NcoI   PflMI   PmeI   PmlI   PpuMI   PshAI   RsrII   SacII   SanDI   SexAI   SfiI   SgfI   SrfI   Tth111I   XcmI
 

 

THIS PRODUCT IS INTENDED FOR RESEARCH PURPOSES ONLY

CAUTION: Not intended for human or animal diagnostic or therapeutic uses.

Limited Warranty

ParaTechs warrants that, at the time of shipment, the Product will conform to the specifications that accompany the Product. This warranty limits ParaTechs liability to replacement of the Product. PARATECHS MAKES NO OTHER WARRANTIES, EXPRESS OR IMPLIED, WITH RESPECT TO THE PRODUCT; INCLUDING ANY WARRANTIES OF MECHANTABILITY OR FITNESS FOR ANY PARTICULAR PURPOSE OR THAT THE PRODUCT DOES NOT INFRINGE ANY PROPRIETARY RIGHTS OF ANY THIRD PARTY.
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pAcVE.02 Instructions

Instructions for Product No.: 20020 Qty: 10µg

Description

pAcVE.02 is a Vankyrin-Enhanced (VE) baculovirus transfer vector designed for high levels of recombinant gene expression under the control of the Autographa californica nucleopolyhedro virus (AcMNPV) polyhedrin promoter. pAcVE.02 is derived from pUC57 and contains the pUC origin of replication and the ampicillin resistance gene for propagation and selection in bacteria. pAcVE.02 is designed for the direct secretion of recombinant proteins utilizing the honeybee melittin signal sequence (Tessier et al., 1991). An N-terminal 8xHis-tag is provided for ease of purification. Cloning into pAcVE.02 requires the DNA insert to be in frame with the N-terminal 8xHis tag. Foreign genes may be cloned into the multiple cloning site utilizing the following restriction sites: NotI; SbfI and NheI. PmeI can be used if a C-terminal 8xHis-tag instead of the N-terminal His-tag is required or if the His-tag is not desired. Increased recombinant protein production over conventional baculovirus transfer vectors is achieved by the co-expression of the gene of interest with a vankyrin expression cassette (Fath-Goodin et al., 2006). pAcVE.02 contains ORF 603 and ORF1629 sequences and allows recombination with the viral DNA for insertion into the polyhedrin locus. The baculovirus transfer vector is compatible with any baculovirus system that utilizes homologous recombination in insect cells, including flashBAC (Oxford Expression Technologies) and Bac-N-Blue (Invitrogen).

Contents

The plasmid DNA is dissolved in TE buffer (10mM Tris-HCl, 1 mM EDTA pH7.5). pAcVE.02 baculovirus transfer vector is provided at 10µg in 50µl.

Storage

The transfer vector should be placed at -20º C for long-term storage.

Handling

For expression of recombinant protein under the polyhedrin promoter, the gene of interest should be ligated into an appropriate restriction site in the multiple cloning site of the pAcVE.02 vector and should be in frame with the N-terminal His-tag (see diagram). Transform the recombinant pAcVE.02 transfer vector in E. coli cells (DH5α, Top10 or any other suitable strain), propagate the cells under ampicillin selection and purify the recombinant plasmid using standard plasmid purification protocols. For construction of recombinant AcMNPV perform a co-transfection of the purified recombinant pAcVE.02 vector with linearized baculovirus DNA suitable for homologous recombination in insect cells.

pAcVE.02 Vector Map and Multiple Cloning Site

Unique Restriction Sites

AgeI   AleI   AlwNI   ApaI   AvaI   AvrII   BamHI   BbvCI   BclI   BmgBI   BmtI   BplI   BpmI   Bpu10I   BsaAI   BsaBI   BseRI   BseYI   BspMI   BstAPI   BstBI   BstXI   EagI   Eco53kI   EcoNI   EcoRI   HindIII   HpaI   KpnI   NaeI   NdeI   NgoMIV   NheI   NotI   NruI   PciI   PfoI   PmeI   PpuMI   PspOMI   SacI   SapI   SbfI   SgrAI   SmaI   SnaBI   SpeI   SphI   StuI   StyI   SwaI   XbaI   XmaI

Absent Restriction Sites

AscI   AsiSI   BglII   BlpI   BsiWI   BspEI   BssHII   BstEII   BstZ17I   Bsu36I   BtgI   DraIII   FseI   FspAI   MscI   NcoI   PflMI   PmlI   PshAI   RsrII   SacII   SanDI   SexAI   SfiI   Tth111I   XcmI   XhoI
 

 

THIS PRODUCT IS INTENDED FOR RESEARCH PURPOSES ONLY

CAUTION: Not intended for human or animal diagnostic or therapeutic uses.

Limited Warranty

ParaTechs warrants that, at the time of shipment, the Product will conform to the specifications that accompany the Product. This warranty limits ParaTechs liability to replacement of the Product. PARATECHS MAKES NO OTHER WARRANTIES, EXPRESS OR IMPLIED, WITH RESPECT TO THE PRODUCT; INCLUDING ANY WARRANTIES OF MECHANTABILITY OR FITNESS FOR ANY PARTICULAR PURPOSE OR THAT THE PRODUCT DOES NOT INFRINGE ANY PROPRIETARY RIGHTS OF ANY THIRD PARTY.
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pAcVE.03 Instructions

Instructions for Product No.: 20030 Qty: 10µg

Description

pAcVE.03 is a Vankyrin-Enhanced (VE) baculovirus transfer vector designed for high levels of recombinant gene expression under the control of the Autographa californica nucleopolyhedro virus (AcMNPV) polyhedrin promoter. pAcVE.03 is derived from pUC57 and contains the pUC origin of replication and the ampicillin resistance gene for propagation and selection in bacteria. pAcVE.03 is designed for the direct secretion of recombinant proteins utilizing the honeybee melittin signal sequence (Tessier et al., 1991). An optional C-terminal 6xHis-tag is provided for ease of purification. Cloning into pAcVE.03 requires the DNA insert to be in frame with the honeybee melittin signal sequence. Foreign genes may be cloned into the multiple cloning site utilizing the following restriction sites: NcoI; SbfI; XhoI; BstZ17I; BglII; SacII and EagI. Increased recombinant protein production over conventional baculovirus transfer vectors is achieved by the co-expression of the gene of interest with a vankyrin expression cassette (Fath-Goodin et al., 2006). pAcVE.03 contains ORF 603 and ORF1629 sequences and allows recombination with the viral DNA for insertion into the polyhedrin locus. The baculovirus transfer vector is compatible with any baculovirus system that utilizes homologous recombination in insect cells, including flashBAC (Oxford Expression Technologies) and Bac-N-Blue (Invitrogen).

Contents

The plasmid DNA is dissolved in TE buffer (10mM Tris-HCl, 1 mM EDTA pH7.5). pAcVE.03 baculovirus transfer vector is provided at 10 µg in 50 µl.

Storage

The transfer vector should be placed at -20º C for long-term storage.

Handling

For expression of recombinant protein under the polyhedrin promoter, the gene of interest should be ligated into an appropriate restriction site in the multiple cloning site of the pAcVE.03 vector and should be in frame with the honey bee melittin signal sequence and the C-terminal 6xHis-tag if desired (see diagram and table). In that instance the stop codon needs to be omitted. Transform the recombinant pAcVE.03 transfer vector in E. coli cells (DH5α, Top10 or any other suitable strain), propagate the cells under ampicillin selection and purify the recombinant plasmid using standard plasmid purification protocols. For construction of recombinant AcMNPV perform a co-transfection of the purified recombinant pAcVE.03 vector with linearized baculovirus DNA suitable for homologous recombination in insect cells.

pAcVE.03 Vector Map and Multiple Cloning Site

Restriction Enzymes Suitable for In-Frame Cloning

Unique Restriction Sites

AgeI   AleI   AlwNI   ApaI   AvrII   BamHI   BbvCI   BclI   BglII   BmgBI   BplI   BpmI   Bpu10I   BsaAI   BsaBI   BseRI   BseYI   BstAPI   BstXI   BstZ17I   EagI   Eco53kI   EcoNI   EcoRI   HindIII   HpaI   KpnI   NaeI   NcoI   NdeI   NgoMIV   NotI   NruI   PciI   PfoI   PpuMI   PspOMI   SacI   SacII   SapI   SbfI   SgrAI   SmaI   SnaBI   SpeI   SphI   StuI   SwaI   XbaI   XhoI   XmaI

Absent Restriction Sites

AscI   AsiSI   BlpI   BmtI   BsiWI   BspEI   BspMI   BssHII   BstEII   Bsu36I   DraIII   FseI   FspAI   MscI   NheI   PflMI   PmeI   PmlI   PshAI   RsrII   SanDI   SexAI   SfiI   Tth111I   XcmI
 

 

THIS PRODUCT IS INTENDED FOR RESEARCH PURPOSES ONLY

CAUTION: Not intended for human or animal diagnostic or therapeutic uses.

Limited Warranty

ParaTechs warrants that, at the time of shipment, the Product will conform to the specifications that accompany the Product. This warranty limits ParaTechs liability to replacement of the Product. PARATECHS MAKES NO OTHER WARRANTIES, EXPRESS OR IMPLIED, WITH RESPECT TO THE PRODUCT; INCLUDING ANY WARRANTIES OF MECHANTABILITY OR FITNESS FOR ANY PARTICULAR PURPOSE OR THAT THE PRODUCT DOES NOT INFRINGE ANY PROPRIETARY RIGHTS OF ANY THIRD PARTY.
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VE-BEVS Frequently Asked Questions

1. What cell line were the VE-cell lines derived from?
2. Do I need to keep the VE-cell lines under antibiotic selection to prevent loss of expression of the vankyrin protein?
3. What is the average size of the VE-cells?
4. What media are VE-cells frozen in?
5. How many passages may I maintain my cells?
6. How do I start my VE-cell lines?
7. How do I freeze my cells down?
8. Which VE-cell line should I purchase?

1. What cell line were the VE-cell lines derived from?

The VE-cell lines are engineered from Sf9 cells that were originally derived from the ovarian tissue of the fall armyworm (Spodoptera frugiperda).

2. Do I need to keep the VE-cell lines under antibiotic selection to prevent loss of expression of the vankyrin protein?

No, the VE-cell lines are stably transformed cell lines and antibiotic treatment is not needed to maintain vankyrin protein expression.

3. What is the average size of the VE-cells?

While derived from Sf9 cells the VE-cells are larger in size than the parental Sf9 cells. VE-cells are approximately 21 microns in diameter.

4. What media are VE-cells frozen in?

Cells were frozen in 50% fresh Sf-900 II serum free medium, 50% conditioned Sf-900 II serum free medium and Dimethyl Sulfoxide (DMSO) to a final concentration of 7.5%

5. How many passages may I maintain my cells?

We recommend starting new cells after they have been through 30 passages. A passage is considered anytime you add media to dilute your cells.

6. How do I start my VE-cell lines?

Thawing Cells:

Thaw frozen cells rapidly in a 37°C water bath. Decontaminate the outside of the vial with 70% ethanol before transferring the cells into one T-25 cm² flask. We recommend starting the cells in adherent culture and then adapting to shaker culture after 2 passages.

Adherent Culture: Add 1 ml of Sf-900 II serum-free medium (SFM) into the cryovial containing thawed cells. Gently resuspend the cells. Remove the cells from the cryovial and transfer into one T25 flask containing 5 mL of medium. Transfer flasks to a 270C incubator and allow the cells to attach for 45-60 minutes before replacing the medium with 5 mL fresh Sf-900 II SFM. Subculture cells when they have reached a density of >80% confluency. Release cells from the flask’s surface by tapping the flask sharply against your palm until > 75% of the cells have detached and transfer 2 mL cells into a new T25 flask containing 3 mL of medium.

Suspension Culture: To start a suspension culture, release the cells from two T25 monolayer cultures and transfer the entire volume from one flask and 3 mL from the second flask (a total volume of 8 ml) into a 125 mL shaker flask containing12 mL of fresh Sf-900 II SFM. Use the remaining 2 mL of cells to continue the cell line as adherent culture in a T-25 flask.

Incubate Erlenmeyer flask in a 27ºC incubator on an orbital shaker platform rotating at 100-110 rpm. Loosen caps of flasks to allow proper oxygenation/aeration. Allow the cells to grow for 3-4 days. Count the cells from the starter flask and transfer the volume of cells necessary to reach a seeding density of 1×106 cells/mL in 50 mL of Sf-900 II SFM in a 125 mL shaker flask. Once a suspension culture has been established and a cell density of 5-8×106 viable cells/mL has been reached VE cells are routinely diluted to a cell density of 0.8-1×106 viable cells/mL with Sf-900 II SFM.

7. How do I freeze my cells down?

Freeze cells at a density of >2×107 viable cells/mL in a freezing medium composed of 50% fresh Sf-900 II serum free medium (SFM), 50% conditioned Sf-900 II serum free medium and Dimethyl Sulfoxide (DMSO) to a final concentration of 7.5% (optional freezing media: fresh Sf-900 II SFM (Invitrogen™), 10% heat-inactivated FBS and DMSO to a final concentration of 7.5%). Centrifuge cells at 100g at 4°C for 5-10 minutes, remove the supernatant and re-suspend the pellet in an appropriate volume of chilled freezing medium to reach a density of >2×107 viable cells/mL. Transfer suspension into a cryovial. Place cells in a styrofoam container and place at -20°C for one hour, then transfer the styrofoam container with cells to -80°C overnight before transferring the cells to liquid nitrogen (vapor phase). Frozen cells remain viable if properly stored in liquid nitrogen.

8. Which VE-cell line should I purchase?

VE-CL-01 (order #10010) displays the most prolonged enhanced protein expression (up to 7 days post-infection) when using conventional BEVS viruses. We recommend VE-CL-01 for laboratories working with highly stable intracellular and secreted proteins as the pronounced enhancement of viability in these cells allows for prolonged expression and accumulation of recombinant protein over time.

VE-CL-02 (order # 10020) displays a sharp peak of enhanced protein expression at day 3 and day 4 post-infection. We recommend VE-CL-02 for laboratories working with highly unstable or toxic proteins as enhanced protein expression occurs at a very high level and over a very short time interval in these cells.

VE-CL-03 (order # 10030) displays an intermediate phenotype to VE-CL-01 and VE-CL-02 showing significantly enhanced protein expression and moderately enhanced longevity. Due to its intermediate expression properties, we recommend VE-CL-03 for laboratories working with proteins of unknown toxicity and stability or for general enhancement of most recombinant proteins expressed in conventional recombinant BEVS viruses.

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VE-BEVS Publications

Fath-Goodin A, Kroemer JA, Webb BA (2009). The Campoletis sonorensis ichnovirus vankyrin protein P-vank-1 inhibits apoptosis in insect Sf9 cells. Insect Mol Biol. 18(4):497-506. PMID: 19453763

Fath-Goodin A, Kroemer J, Martin S, Reeves K, Webb BA (2006). Polydnavirus genes that enhance the baculovirus expression vector system. Adv Virus Res. 68:75-90. Review. PMID: 16997009

Kroemer JA, Webb BA (2006). Divergences in protein activity and cellular localization within the Campoletis sonorensis Ichnovirus Vankyrin family. J Virol. 80(24): 12219-28. PMID: 1700565

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VE-BEVS Presentations

Steele K, oral presentation entitled “Expression of mammalian glycoproteins and other difficult to express proteins with the vankyrin-enhanced baculovirus expression system.” Presented during PEGS: The Essential Protein Engineering Summit, Boston, MA, May 6, 2014. [PDF]

Steele K, poster presentation entitled “End the poor protein yield crisis by delaying cell lysis: new baculovirus transfer vectors encoding the anti-apoptotic vankyrin protein.” Presented during the International Society for BioProcess Technology, 4th Spring meeting, Washington D.C., March 12, 2014

Oral presentation entitled “Function and Versatility of Vankyrin Gene-Mediated Enhancement of Baculovirus Expression Vector Protein Production” at ISBiotech 2nd Annual Meeting “Baculovirus Expression Technology”, Rosslyn, VA, April 2-5, 2012

Poster presentation entitled “Log-Scale Improvement in Protein Yield with Vankyrin-Enhanced Transfer Vector” at the Cambridge Healthtech Institute “Baculovirus Technology” conference, Boston, MA, August 22-23, 2011 [PDF]

Oral presentation entitled “Expanding the Utility of Viral Ankyrin Genes in Baculoviruses” at the Wilbio 12th International Conference on Baculovirus and Insect Cell Culture, San Antonia, TX, February 2-4, 2009

Oral presentation entitled “Improved Recombinant Protein Production by ParaTechs’ Vankyrin-Enhanced Baculovirus Expression Technology” ” at the Cambridge Healthtech Institute “Baculovirus Technology” conference, Boston, MA, August 2008

Poster presentation entitled “Enhanced Recombinant Protein Production by ParaTechs’ Vankyrin Enhanced Baculovirus Expression Technology” at the Wilbio 11th International Conference on Baculovirus and Insect Cell Culture, Seattle, WA, February 25-27, 2008 [PDF]

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VE-BEVS Licensing

The VE-BEVS Cell Line (“Product”) and VE-BEVS Transfer Vectors (“Product”) were developed in collaboration by scientists at ParaTechs and the University of Kentucky Lexington for expression of recombinant proteins. One or more patents or patent applications owned by the University of Kentucky Lexington cover components of the Product.

ParaTechs Corporation has an exclusive license to sell the Product to scientists for academic research or one year commercial evaluation only, under the terms described below. Use of the Product for any Commercial Purpose (as defined below) other than evaluation requires the user to obtain a commercial license as detailed below. Before using the Product, please read the terms and conditions set forth below. Your use of the Product shall constitute acknowledgement and acceptance of these terms and conditions. If you do not wish to use the Product pursuant to these terms and conditions, please contact ParaTechs Technical Service Department to return the unused and unopened Product for full credit.

ParaTechs grants the purchaser a non-exclusive license to use the enclosed Product for academic research or for commercial evaluation purposes only. The Product is being transferred to you in furtherance of, and reliance on, such license. You may not use the Product, or the materials contained therein, for any Commercial Purpose without a license for such purpose from ParaTechs Corporation.

Commercial Purpose include any use of Product in a Commercial Product, the manufacture of a Commercial Product, any resale of the Product, any use (other than evaluation) of Product to facilitate or advance research or development of a Commercial Product, and any use (other than evaluation) of the Product to facilitate or advance any research or development program the results of which will be applied to the development of Commercial Products.

“Commercial Product” means any product intended for commercial use.

Access to the Product must be limited solely to those officers, employees and students of your entity who need access to perform the aforementioned research or evaluation. Each such officer, employee and student must be informed of these terms and conditions and agree in writing, to be bound by same. You may not distribute the Product to others. You may not transfer modified, altered, or original material from the Product to a third party without written notification to and written approval from ParaTechs.

Inquiries for commercial use should be directed to agoodin@paratechs.com.

Patent information:
VE-BEVS Cell Line United States Patent 7,842,493 [PDF]
VE-BEVS Transfer Vector United States Patent 7,629,160 [PDF]

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