3. Baculovirus stock P2 (e.g., AcMNPV-Orf2-PCV2).
4. 5 mL sterile syringe.
5. Luer lock lids.
6. Materials for the cultivation of insect cells in a stirred-tank
bioreactor (see Subheading 2.4.5).
7. Materials for online dielectric spectroscopy (see Subheading
2.4.6).
8. Materials for cell counting (see Subheading 2.4.2).
2.7.2 Materials
for the Harvesting
of the Produced
Recombinant Proteins
1. Glass bottles with connector cap for harvesting (e.g., the
connected inoculation bottle, see Subheadings 2.4.5 and
3.7.2).
2. Protease inhibitor (e.g., Protease Inhibitor Cocktail, VWR,
Radnor, Pennsylvania, USA).
3. Sterile single-use pipette tips (e.g., serological pipettes, Sarstedt, Nu ¨mbrecht, Germany).
4. Pipette holder/rubber bulb (e.g., pipetus, Hirschmann Laborger€ ate, Eberstadt, Germany).
5. Centrifuge tubes (e.g., Sarstedt, Nu ¨mbrecht, Germany).
6. Centrifuge (e.g., 6-16 KS, Sigma-Aldrich, St. Louis,
Missouri, USA).
7. À80
C freezer.
3 Methods
3.1 Transfer Cloning
Vector
The DNA sequence of the desired gene which encodes for the
protein of interest must be known, and can either be synthesized
de novo, or amplified by PCR. The desired target sequence must
then be integrated into an acceptor vector. Depending on which
Baculovirus Expression Vector System is used, different acceptor
vectors are available. To insert the desired gene sequence into the
acceptor vector, Golden Gate cloning, or classical restriction ligation cloning, can be used. Other genetic modifications, such as a
lacZα cassette (which is assumed in this case), a reporter gene, or
others, should be considered too (see Note 7).
3.2 Generation
of Recombinant
Baculoviral DNA
This chapter describes the methods for generating baculoviral DNA
(also called bacmid). The protocol corresponds, apart from a few
deviations, to the Bac-to-Bac Manual of Life Technologies [6].
3.2.1 Transformation
of Competent MAX
Efficiency
® DH10-Bac™
E. coli
1. Prepare agar plates containing kanamycin, tetracycline, X-Gal,
and IPTG for the selection of positive E. coli transformants.
2. Thaw one vial competent MAX Efficiency
® DH10-Bac™
on ice.
106
Julie Harnischfeger et al.
4. 5 mL sterile syringe.
5. Luer lock lids.
6. Materials for the cultivation of insect cells in a stirred-tank
bioreactor (see Subheading 2.4.5).
7. Materials for online dielectric spectroscopy (see Subheading
2.4.6).
8. Materials for cell counting (see Subheading 2.4.2).
2.7.2 Materials
for the Harvesting
of the Produced
Recombinant Proteins
1. Glass bottles with connector cap for harvesting (e.g., the
connected inoculation bottle, see Subheadings 2.4.5 and
3.7.2).
2. Protease inhibitor (e.g., Protease Inhibitor Cocktail, VWR,
Radnor, Pennsylvania, USA).
3. Sterile single-use pipette tips (e.g., serological pipettes, Sarstedt, Nu ¨mbrecht, Germany).
4. Pipette holder/rubber bulb (e.g., pipetus, Hirschmann Laborger€ ate, Eberstadt, Germany).
5. Centrifuge tubes (e.g., Sarstedt, Nu ¨mbrecht, Germany).
6. Centrifuge (e.g., 6-16 KS, Sigma-Aldrich, St. Louis,
Missouri, USA).
7. À80
C freezer.
3 Methods
3.1 Transfer Cloning
Vector
The DNA sequence of the desired gene which encodes for the
protein of interest must be known, and can either be synthesized
de novo, or amplified by PCR. The desired target sequence must
then be integrated into an acceptor vector. Depending on which
Baculovirus Expression Vector System is used, different acceptor
vectors are available. To insert the desired gene sequence into the
acceptor vector, Golden Gate cloning, or classical restriction ligation cloning, can be used. Other genetic modifications, such as a
lacZα cassette (which is assumed in this case), a reporter gene, or
others, should be considered too (see Note 7).
3.2 Generation
of Recombinant
Baculoviral DNA
This chapter describes the methods for generating baculoviral DNA
(also called bacmid). The protocol corresponds, apart from a few
deviations, to the Bac-to-Bac Manual of Life Technologies [6].
3.2.1 Transformation
of Competent MAX
Efficiency
® DH10-Bac™
E. coli
1. Prepare agar plates containing kanamycin, tetracycline, X-Gal,
and IPTG for the selection of positive E. coli transformants.
2. Thaw one vial competent MAX Efficiency
® DH10-Bac™
on ice.
106
Julie Harnischfeger et al.