pbluebac4.5 A Baculovirus Transfer Vector Catalog no. V Version D January 27,

Transcription

1 pbluebac4.5 A Baculovirus Transfer Vector Catalog no. V Version D January 27, tech_service@invitrogen.com

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3 Table of Contents Table of Contents... iii Important Information...iv Introduction...1 Overview...1 Methods...2 Cloning into pbluebac Guidelines for Isolating Recombinant Virus...4 Appendix...6 pbluebac4.5 Vector...6 pbluebac4.5/cat...8 Technical Service...9 References...11 iii

4 Important Information Contents 20 µg pbluebac4.5, lyophilized 20 µg pbluebac4.5/cat, lyophilized Shipping and Storage Lyophilized pbluebac4.5 and pbluebac4.5/cat are shipped at room temperature and stored at -20 C. Limited Use Label License No. 69: Baculovirus Vectors and Reagents This recombinant baculovirus expression system is the subject of one ore more of US patents 4,745,051; 4,879,236; 5,155,037; and 5,278,050 and corresponding foreign applications licensed to Invitrogen Corporation and sold for research purposes only. Utilization of this product or system for the expression of gene products for commercial product development, manufacturing, or sale requires a license under the rights of The Texas A&M University System. Please contact: Technology Licensing Manager, Agriculture and Life Sciences, Technology Licensing Office, The Texas A&M University System, 310 Wisenbaker, College Station, TX Phone: (409) ; Fax: (409) You may not distribute the System or the vectors or host strains contained in it to others. You may not transfer modified, altered, or original material from the System to a third party without written notification to, and written approval from Invitrogen. You may not assign, sub-license, rent, lease or otherwise transfer any of the rights or obligations set forth herein, except as expressly permitted by Invitrogen. Product Qualification The vectors are qualified by restriction digestion analysis. Refer to the table below for the enzymes used and the expected fragments. Restriction Enzyme pbluebac4.5 pbluebac4.5/cat Ase I 3591, 1349 bp 4378, 1349 bp BamH I 4940 bp 5727 bp Hind III 4940 bp 4940, 787 bp iv

5 Introduction Overview Introduction pbluebac4.5 is a baculovirus transfer vector used for expression of full-length genes that contain an ATG translation initiation codon. The vector is derived from pjvetl-z (Vialard, et al., 1990) and contains the early-to-late (ETL) promoter and the polyhedrin promoter from AcMNPV. The ETL promoter directs the synthesis of β-galactosidase (Crawford and Miller, 1988) while the polyhedrin promoter controls the synthesis of foreign gene products. The 5 polyhedrin mrna leader sequence of the baculovirus transfer vector pvl941 is utilized. The native polyhedrin ATG was removed through site directed mutagenesis. The modified expression vector recombines with Invitrogen's Bac-N-Blue DNA (Catalog no. K855-01) to yield recombinants which are occ -, produce foreign gene products, and form blue plaques when 5-bromo-4-chloro-3-indolyl-β-D-galactosidase (X-gal) or a derivative is present in the agarose overlay. All known translational regulatory sequences downstream and upstream of the native ATG of the polyhedrin gene were conserved. Recombination With Bac-N- Blue DNA pbluebac4.5 contains the 5 portion of the lacz gene and ORF1629. Recombination occurs between these sequences and lacz and ORF1629 sequences in Bac-N-Blue DNA, forming blue, recombinant plaques on medium containing X-gal. Bac-N-Blue DNA Bsu36 I Bsu36 I P 603 P 1629 ORF603 3 lacz 5 ORF1629 P ETL P PH P lacz Gene of Interest ORF1629 pbluebac4.5 Important pbluebac4.5 can only be used with Bac-N-Blue DNA (Catalog no. K855-01). The vector cannot be used with Invitrogen's original linear AcMNPV DNA, BaculoGold (PharMingen) or BacPAK6 (Clontech) AcMNPV DNA. Because the vector does not contain ORF603, and the lacz sequences found in other linear DNAs are in the opposite orientation, recombination will not occur properly. 1

6 Methods Cloning into pbluebac4.5 General Molecular Biology Techniques For help with DNA ligations, E. coli transformations, restriction enzyme analysis, DNA sequencing, and DNA biochemistry, see Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989)or Current Protocols in Molecular Biology (Ausubel, et al., 1994). Maintenance of pbluebac4.5 In order to propagate and maintain pbluebac4.5, we recommend that you take the vector and resuspend the lyophilized material in 20 µl sterile water to make a 1 µg/ µl stock solution. Store at -20 C. Use this stock solution to transform a reca, enda E. coli strain like TOP10, TOP10F, DH5α, JM109, or equivalent. Transformants are selected on LB plates containing µg/ml ampicillin. The production of nonfused proteins requires DNA inserts containing a translation initiation ATG. Generally, transfer vectors which contain intact polyhedrin leader sequences (pbluebac4.5, pvl1392, pvl1393) may yield higher levels of expression than vectors which contain interrupted leader sequences. However, protein translation may initiate at the mutated ATG (ATT) if the recombinant gene is inserted in frame with the polyhedrin open reading frame. This may result in two recombinant expression products: the nonfused recombinant protein (as the major expression product), and the recombinant protein fused with polyhedrin (Beames, et al., 1991). continued on next page 2

7 Cloning into pbluebac4.5, Continued Multiple Cloning Site of pbluebac Below is the multiple cloning site for pbluebac4.5. Note that the multiple cloning site has been modified from pbluebac4 to eliminate an Nco I site that contained an ATG. In addition, unique Sma I and Xba I sites have been added between the Kpn I and EcoR I sites. Restriction sites are labeled to indicate the cleavage site. The multiple cloning site has been confirmed by sequencing and functional testing. Polyhedrin Forward Sequencing Priming Site Start of Transcription GATATCATGG AGATAATTAA AATGATAACC ATCTCGCAAA TAAATAAGTA Baculovirus Forward PCR Priming Site wild-type ATG mutated to ATT TTTTACTGTT TTCGTAACAG TTTTGTAATA AAAAAACCTA TAAATATTCC Nhe I Bam H I Xho I Sac I GGATTATTCA TACCGTCCCA CCATCGGGCG TGCTAGCGGA TCCGAGCTCG Bgl II Pst I Kpn I Sma IXba I EcoR I BstB I Hind III Sal I AGATCTGCAG CTGGTACCCG GGTCTAGAAT TCGAAGCTTG GAGTCGACAA CTTGTTTATT GCAGCTTATA ATGGTTACAA ATAAAGCAAT AGCATCACAA SV40 polyadenylation sequence ATTTCACAAA TAAAGCATTT TTTTCACTGC ATTCTAGTTG TGGTTTGTCC AAACTCATCA ATGTATCTTA TCATCTCTCG ACTCTGCTGA AGAGGAGGAA 351 ATTCTCCTTG AAGTTTCCCT GGTGTTCAAA GTAAAGGAGT TTGCACCAGA CGCACCTCTG TTCACTGGTC CGGCGTATTA AAACACGATA CATTGTTATT Baculovirus Reverse PCR Priming Site AGTACATTTA TTAAGCGCTA GATTCTGTGC GTTGTTGATT TACAGACAAT 3

8 Guidelines for Isolating Recombinant Virus Introduction The following guidelines and recommendations are provided for your convenience. If you need more details about the techniques discussed, refer to Current Protocols in Molecular Biology, Unit (Ausubel, et al., 1994), The Baculovirus Expression System: A Laboratory Guide (King and Possee, 1992), or Baculovirus Expression Vectors: A Laboratory Manual (O'Reilly, et al., 1992). E. coli Transformation Transform your ligation mixtures into a competent reca, enda E. coli strain (e. g. TOP10, DH5α) and select on LB plates containing µg/ml ampicillin. Select clones and analyze for the presence and orientation of your insert. RECOMMENDATION We recommend that you sequence your construct using the Polyhedrin Forward and appropriate reverse primer sequences to confirm that your gene is in the correct orientation, if desired. Refer to the diagram on the previous page for the sequence and location of the primer binding sites. For your convenience, Invitrogen offers a custom primer synthesis service. For more information, refer to our Web site ( or contact Technical Service (see page 9). Transfection into Insect Cells In addition to your construct in pbluebac4.5, we recommend that you include the control vector pbluebac4.5/cat as a positive control for expression. Plasmid DNA for transfection into insect cells must be very clean and free from phenol and sodium chloride. Contaminants will kill the cells and, in addition, salt will interfere with the Cellfectin, decreasing transfection efficiency. We recommend purifying plasmid DNA for transfection using resin-based DNA purification methods (i.e. S.N.A.P. MidiPrep Kit, Catalog no. K ). For methods to transfect insect cells, screen for recombinant plaques, PCR confirmation of recombinant plaques, and expression, see the Bac-N-Blue Transfection and Expression Guide or the references listed above. This manual is available on our Web site at or contact Technical Service (see page 9). Designing PCR Primers To perform PCR analysis (see next page), you will need to design appropriate PCR primers to allow verification of recombinant viruses containing inserts. We recommend designing forward and reverse primers with the following sequence: Baculovirus Forward primer: 5 -TTTACTGTTTTCGTAACAGTTTTG-3 Baculovirus Reverse primer: 5 -CAACAACGCACAGAATCTAGC-3 These primers flank the polyhedrin region and are compatible with all polyhedrin promoter based baculovirus transfer vectors. Refer to the diagram on page 3 to locate the primer binding site for the vector. Note: These are suggested primer sequences, and are not available from Invitrogen. If you would like information on placing custom primer orders, visit our Web site ( or contact Technical Service (see page 9). continued on next page 4

9 Guidelines for Isolating Recombinant Virus, Continued PCR Analysis If you use PCR to analyze putative recombinant plaques in order to isolate a pure clone, you will need to know the expected size of your PCR fragment. Using the Baculovirus PCR Primers described on the previous page, the following PCR products are generated: Vector PCR Product (bp) pbluebac pbluebac4.5/cat 1222 Add the size of the PCR product from the pbluebac4.5 vector alone to the size of the expected PCR product. For example, if you have a 1000 bp fragment cloned into the Pst I site of pbluebac4.5, the expected size of your PCR fragment will be 1435 bp. pbluebac4.5/cat Control Vector The pbluebac4.5/cat vector is included as a positive control for expression in insect cell lines. In order to test for expression, you will need to co-transfect this vector with Bac-N-Blue AcMNPV DNA to generate recombinant virus. To do this, you will need to perform the following steps: Step Action 1 Co-transfect pbluebac4.5/cat and Bac-N-Blue DNA into Sf9 cells 2 Identify blue, recombinant plaques and purify using the Plaque Assay 3 Verify isolation of pure, recombinant virus by PCR (see page 7) 4 Generate a high-titer stock for expression experiments 5 Infect a suspension culture of Sf9 or the desired insect cell line and assay for CAT expression Note that you will be able to identify recombinant plaques by plating on medium containing X-gal. Select only the blue plaques for analysis. Transfection and Expression Refer to the Bac-N-Blue Transfection and Expression Guide for procedures to cotransfect, plaque purify, produce high-titer stocks, and express your construct in insect cells. This manual is available on our Web site at or contact Technical Service (see page 9). 5

10 Appendix pbluebac4.5 Vector Description pbluebac4.5 (4940 bp) is a baculovirus transfer vector designed to allow expression of your gene of interest in insect cell lines. Expression of your recombinant protein is driven by the polyhedrin promoter. Features of pbluebac4.5 The important elements of pbluebac4.5 are described in the following table. All features have been functionally tested. Feature Polyhedrin promoter Multiple Cloning Site SV40 polyadenylation signal ORF1629 recombination sequences lacz recombination sequences Baculovirus early-to-late (ETL) promoter Ampicillin resistance gene (β-lactamase) puc origin Benefit Allows efficient, high-level expression of your recombinant protein Allows insertion of your gene Increased transcription termination efficiency and mrna stability (Westwood, et al., 1993) Permits integration of your gene into the Bac-N-Blue DNA and restores the essential ORF1629 for production of viable, recombinant virus Permits integration of your gene into the Bac-N-Blue DNA and production of blue, recombinant plaques for easy selection Allows expression of the intact lacz gene to produce blue, recombinant plaques in insect cells Selection of vector in E. coli High-copy number replication and growth in E. coli continued on next page 6

11 pbluebac4.5 Vector, Continued Map of pbluebac4.5 The figure below summarizes the features of the pbluebac4.5 vector. The complete nucleotide sequence for pbluebac4.5 is available for downloading from our Web site at or by contacting Technical Service (see page 9). Details of the multiple cloning site are shown on page 3. Nhe I BamH I Xho I Sac I Bgl II Pst I Kpn I Sma I Xba I EcoR I BstB I Hind III Sal I 5 lacz Fragment P ETL P PH SV40 pa pbluebac kb Recombination Sequence puc Comments for pbluebac4.5: 4940 nucleotides Ampicillin Polyhedrin promoter (P PH ): bases 7-95 Multiple cloning site: bases SV40 polyadenylation sequence: Recombination sequences (ORF1629): bases Ampicillin resistance gene: bases puc origin: bases lacz fragment: bases (C) lacz sequence homologous to lacz sequence in Bac-N-Blue DNA: bases (C) Early-to-late promoter (P ETL ): bases (C) (C) = complementary strand 7

12 pbluebac4.5/cat Description pbluebac4.5/cat is a 5727 bp control vector containing the gene for chloramphenicol acetyl transferase (CAT). It was constructed by digesting pbluebac4.5 with Hind III and dephosphorylating with calf intestinal alkaline phosphatase (CIAP). A Hind III fragment containing the gene for chloramphenicol acetyl transferase was then ligated into pbluebac4.5. Map of Control Vector The figure below summarizes the features of the pbluebac4.5/cat vector. The complete nucleotide sequence for pbluebac4.5/cat is available for downloading from our Web site at or by contacting Technical Service (see page 9). 5 lacz Fragment P ETL P PH CAT SV40 pa pbluebac4.5/ CAT 5.7 kb Recombination Sequence Comments for pbluebac4.5/cat: 5727 nucleotides puc Polyhedrin promoter (P PH ): bases 7-95 CAT ORF: bases SV40 polyadenylation sequence: Recombination sequences (ORF1629): bases Ampicillin resistance gene: bases puc origin: bases lacz fragment: bases (C) lacz sequence homologous to lacz sequence in Bac-N-Blue DNA: bases (C) Early-to-late promoter (P ETL ): bases (C) (C) = complementary strand Ampicillin 8

13 Technical Service World Wide Web Visit the Invitrogen Web Resource using your World Wide Web browser. At the site, you can: Get the scoop on our hot new products and special product offers View and download vector maps and sequences Download manuals in Adobe Acrobat (PDF) format Explore our catalog with full color graphics Obtain citations for Invitrogen products Request catalog and product literature Once connected to the Internet, launch your Web browser (Internet Explorer 5.0 or newer or Netscape 4.0 or newer), then enter the following location (or URL): the program will connect directly. Click on underlined text or outlined graphics to explore. Don't forget to put a bookmark at our site for easy reference! Contact Us For more information or technical assistance, call, write, fax, or . Additional international offices are listed on our Web page ( Corporate Headquarters: Invitrogen Corporation 1600 Faraday Avenue Carlsbad, CA USA Tel: Tel (Toll Free): Fax: tech_service@invitrogen.com Japanese Headquarters: Invitrogen Japan K.K. Nihonbashi Hama-Cho Park Bldg. 4F , Hama-Cho, Nihonbashi Tel: Fax: jpinfo@invitrogen.com European Headquarters: Invitrogen Ltd 3 Fountain Drive Inchinnan Business Park Paisley PA4 9RF, UK Tel: +44 (0) Tel (Toll Free): Fax: +44 (0) eurotech@invitrogen.com MSDS Requests To request an MSDS, visit our Web site ( and follow the instructions below. 1. On the home page, go to the left-hand column under Technical Resources and select MSDS Requests. 2. Follow instructions on the page and fill out all the required fields. 3. To request additional MSDSs, click the Add Another button. 4. All requests will be faxed unless another method is selected. 5. When you are finished entering information, click the Submit button. Your MSDS will be sent within 24 hours. continued on next page 9

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15 References Citations Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. E., Seidman, J. G. Smith, J. A., and Struhl, K., eds (1994) Current Protocols in Molecular Biology, John Wiley and Sons, Inc., New York, NY. Beames, B., Braunagel, S. C., Summers, M. D., and Lanford, R. E. (1991) Translational Initiation from an AUU Codon of a Baculovirus Vector. BioTechniques 11: Crawford, A. M., and Miller, L. K. (1988) Characterization of an Early Gene Accelerating Expression of Late Genes of the Baculovirus Autographa californica Nuclear Polyhedrosis Virus. J. Virology 62: King, L. A. and Possee, R. D. (1992) The Baculovirus Expression System: A Laboratory Guide. Chapman and Hall. New York, NY. O'Reilly, D. R., Miller, L. K. and Luckow, V. A. (1992) Baculovirus Expression Vectors: A Laboratory Manual. W. H. Freeman and Company. New York, N. Y. Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press. Plainview, New York. Vialard, J., Lalumiere, M., Vernet, T., Briedis, D., Alkhatib, G., Henning, D., Levin, D., and Richardson, C. (1990) Synthesis of the Membrane Fusion and Hemaglutinin Proteins of Measles Virus, using a Novel Baculovirus Vector Containing the β-galactosidase Gene. J. Virol. 64: Westwood, J. A., Jones, I. M., and Bishop, D. H. L. (1993) Analyses of Alternative Poly(A) Signals for Use in Baculovirus Expression Vectors. Virology 195: Invitrogen Corporation. All rights reserved. 11