Bac-to-Bac Baculovirus Expression System

1 USER GUIDEFor Research Use Only. Not for use in diagnostic procedures. Bac-to-Bac Baculovirus Expression System An efficient site-specific transposition System to generate Baculovirus for high-level Expression of recombinant proteins Catalog Numbers 10359-016, 10360-014, 10584-027, 10712-024 Document Part Number 10359 Publication Number MAN0000414 Revision 2 Information in this document is subject to change without notice. DISCLAIMER LIFE TECHNOLOGIES CORPORATION AND/OR ITS AFFILIATE(S) DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE, OR NON-INFRINGEMENT. TO THE EXTENT ALLOWED BY LAW, IN NO EVENT SHALL LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF.

2 IMPORTANT LICENSING INFORMATION These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all applicable Limited Use Label Licenses. INFORMATION FOR EUROPEAN CUSTOMERS The DH10 Bac strain is genetically modified and carries the pBR322-derived plasmid, pMON7124 (bom+, tra , mob ). As a condition of sale, this product must be in accordance with all applicable local legislation and guidelines including EC Directive 90/219/EEC on the contained use of genetically modified organisms. TRADEMARKS The trademarks mentioned herein are the property of Life Technologies Corporation and/or its affiliate(s) or their respective owners. PLURONIC is a registered trademark of BASF Corporation. 2015 Life Technologies Corporation. All rights reserved. 3 Contents About this guide.

3 4 Product Information .. 5 Contents and storage .. 5 Description of the System .. 7 The Bac-to-Bac Baculovirus Expression System .. 10 Experimental outline .. 13 Culturing insect cells .. 14 General guidelines .. 14 Generating the recombinant pFastBac vector .. 16 General information .. 16 Cloning into pFastBac 17 Cloning into pFastBac HT A, B, and C .. 18 Cloning into pFastBac Dual .. 22 Transformation and 24 Generating the recombinant 26 Transforming DH10 Bac E. coli .. 26 Isolating recombinant bacmid DNA .. 30 Analyzing recombinant bacmid DNA by PCR .. 32 Producing recombinant Baculovirus .. 35 Transfecting insect cells .. 35 Isolating P1 viral stock .. 38 Amplifying your baculoviral stock .. 40 Performing a viral plaque assay .. 42 Expressing your recombinant protein .. 49 Troubleshooting .. 52 Appendix A: Support protocols.

4 58 Preparing reagents and media .. 58 Bacmid DNA isolation using PureLink HiPure Maxiprep Kit .. 60 Appendix B: Vectors .. 63 Map and features of pFastBac 1 .. 63 Map and features of pFastBac HT .. 65 Map and features of pFastBac dual .. 67 Map of pFastBac -Gus .. 69 Map of pFastBac HT-CAT .. 70 Map of pFastBac Dual-Gus/CAT .. 71 Appendix C: Ordering information .. 72 Accessory products .. 72 Appendix D: Safety .. 74 Chemical safety .. 74 Biological hazard safety .. 75 Documentation and support .. 76 Obtaining support .. 76 References .. 77 4 About this guide IMPORTANT! Before using this product, read and understand the information in the Safety appendix in this document. Changes from previous version Revision Date Description December 2013 Removed reference to discontinued product: BaculoTiter. Version numbering changed to alphanumeric format and reset to A in conformance with internal document control procedures Purpose of this manual This manual provides an overview of the Bac-to-Bac Baculovirus Expression System , and provides instructions and guidelines to: 1.

5 Clone your gene of interest into the pFastBac donor plasmid of choice. 2. Transform the pFastBac construct into MAX Efficiency DH10 Bac competent E. coli to generate a recombinant bacmid. 3. Transfect the recombinant bacmid DNA into the insect cell line of choice to generate a recombinant Baculovirus . 4. Amplify and titer the baculoviral stock, and use this stock to infect insect cells to express your recombinant protein . 5 Product Information Contents and storage Types of products This manual is supplied with the products listed below. For a list of the reagents supplied with each catalog number, see below and the next page. Product Quantity Cat. no. Bac-to-Bac Baculovirus Expression System 1 kit 10359-016 Bac-to-Bac Vector Kit 1 kit 10360-014 Bac-to-Bac HT Vector Kit 1 kit 10584-027 pFastBac Dual Vector Kit 1 kit 10712-024 Kit components Each catalog number contains the components listed below.

6 IMPORTANT! Note that catalog numbers 10360-014, 10584-027, and 10712-024 contain pFastBac vectors only. See the next page for a detailed description about the specific pFastBac vector and other reagents supplied with each catalog number. Component Cat. no. 10359-016 Cat. no. 10360-014 Cat. no. 10584-027 Cat. no. 10712-024pFastBac Vectors 9 9 9 9 MAX Efficiency DH10 Bac Competent E. coli 9 Cellfectin II Reagent 9 Shipping and storage The Bac-to-Bac Baculovirus Expression System is shipped in three boxes as described below. Upon receipt, store each box as detailed below. All reagents are guaranteed for six months if stored properly. Box Item Shipping Storage 1 pFastBac Vectors Blue ice 2 C to 8 C 2 MAX Efficiency DH10 Bac Competent E. coli Dry ice 85 C to 68 C3 Cellfectin II Reagent Blue ice 2 C to 8 C Vector kits Cat.

7 Nos. 10360-014, 10584-027, and 10712-024 are shipped on blue ice. Upon receipt, store the vectors at 2 C to 8 on next page 6 Contents and storage, continued pFastBac vectors Each catalog number includes a specific pFastBac vector(s) and a corresponding Expression control, and are supplied as detailed below. Store at 2 C to 8 C. Product Cat. no. pFastBac vector Expression control Bac-to-Bac Baculovirus Expression System 10359-016 pFastBac 1 Supplied: 20 L at g/ L in TE, pH * (10 g total) pFastBac -Gus Supplied: 20 L at ng/ L in TE, pH (4 ng total) Bac-to-Bac Vector Kit 10360-014 pFastBac 1 Supplied: 20 L at g/ L in TE, pH (10 g total) pFastBac -Gus Supplied: 20 L at ng/ L in TE, pH (4 ng total) Bac-to-Bac HT Vector Kit 10584-027 pFastBac HT A pFastBac HT B pFastBac HT C Supplied: 20 L each at g/ L in TE, pH (10 g total of each vector) pFastBac HT-CAT Supplied: 15 L at 1 ng/ L in TE, pH (15 ng total) pFastBac Dual 10712-024 pFastBac Dual Supplied: 20 L at g/ L in TE, pH (10 g total) pFastBac Dual-Gus/CAT Supplied: 20 L at ng/ L in TE, pH (4 ng total) *TE buffer, pH.

8 10 mM Tris-HCl, 1 mM EDTA, pH MAX Efficiency DH10 Bac Competent E. coli reagents MAX Efficiency DH10 Bac Chemically Competent E. coli are supplied with the Bac-to-Bac Baculovirus Expression System only, and include the following items. Transformation efficiency is 1 108 cfu/ g DNA. Store at 85 C to 68 C. Item Composition Amount MAX Efficiency Chemically Competent DH10 Bac 5 100 L pUC19 Control DNA 10 pg/ L in 5 mM Tris-HCl, mM EDTA, pH 8 100 L Genotype of DH10 Bac F mcrA (mrr-hsdRMS-mcrBC) 80lacZ M15 lacX74 recA1 endA1 araD139 (ara, leu)7697 galU galK rpsL nupG/bMON14272/pMON7124 Cellfectin II Transfection Reagent Cellfectin II Reagent is supplied with the Bac-to-Bac Baculovirus Expression System only.

9 Amount supplied: 1 mL Composition: 1 mg/mL transfection reagent in membrane-filtered water Storage conditions: 2 C to 8 C 7 Description of the System Bac-to-Bac Baculovirus Expression System The Bac-to-Bac Baculovirus Expression System provides a rapid and efficient method to generate recombinant baculoviruses (Ciccarone et al., 1997). This method was developed by researchers at Monsanto, and is based on site-specific transposition of an Expression cassette into a Baculovirus shuttle vector (bacmid) propagated in E. coli (Luckow et al., 1993). The major components of the Bac-to-Bac Baculovirus Expression System include: A choice of pFastBac donor plasmids that allow generation of an Expression construct containing the gene of interest where Expression of the gene of interest is controlled by a Baculovirus -specific promoter. An E.

10 Coli host strain, DH10 Bac , that contains a Baculovirus shuttle vector (bacmid) and a helper plasmid, and allows generation of a recombinant bacmid following transposition of the pFastBac Expression construct. A control Expression plasmid containing the Gus and/or CAT gene that allows production of a recombinant Baculovirus which, when used to infect insect cells, expresses -glucuronidase and/or chloramphenicol acetyl-transferase. Advantages of the Bac-to-Bac Baculovirus Expression System Using the Bac-to-Bac Baculovirus Expression System to generate a recombinant Baculovirus provides the following advantages over the traditional method using homologous recombination: Requires less than 2 weeks to identify and purify a recombinant Baculovirus as compared to the 4 6 weeks required to generate a recombinant Baculovirus using homologous recombination Reduces the need for multiple rounds of plaque purification as the recombinant virus DNA isolated from selected colonies is not mixed with parental, non-recombinant virus Permits rapid and simultaneous isolation of multiple recombinant baculoviruses, and is suited for the Expression of protein variants for structure/function studies Continued on next page 8 Description of the System , continued Choosing a pFastBac vector A number of pFastBac vectors are available for use with the Bac-to-Bac Baculovirus Expression System (see table below).