Analysis of Laminin Structure and Function with Recombinant Glycoprotein Expressed in Insect Cells - Methods in Molecular Biology: Extracellular Matrix Protocols - page 31

Table 1
Time Requirements for Baculovirus Expression

Week 1 Design and construction of recombinant in baculoviral vector

Week 2 Sf9 cell culture, transfection, and isolation of P 0

Week 3-4 Recombinant baculovirus cloning and expansion

Week 5 Preparation of clonal recombinant baculovirus stocks

Week 6 Infection for large-scale protein production

Week 7 Protein purification is complete

7. After 5 d collect conditioned medium, and remove cell debris by centrifugation

of (2000 g , 10 min, room temperature). The medium should be labeled P 0 and
stored at 4 ° C ( see Note 11 ).

3.2. Recombinant Baculovirus Expansion and Clonal Selection
8. Add 100 10 7 adherent cells in

10 mL medium. Incubate as in step 7 and label the cleared medium P 1 ( see Note 12 ).

µ L P 0 to a 75-cm 2 -flask previously seeded with 1 ×

10 3 cells/well).
The volume of each well should not be <100 mL. Assuming a titer of 10 8 viral
particles/mL for P 1 , infect the cells with 10, 5, 1, 0.5, and 0.1 viral particles/well
on separate plates by dilution of P 1 in medium and adding directly to the plates
( see Note 13 ). Incubate at 27

9. Seed several 96-well plates with Sf9 cells at half maximal density (5 ×

° C for 3 d.

10. Score the wells on each plate for signs of infection ( see Note 12 ). Screen for

recombinant protein by Western dot blot using half the volume of each well (<50 µ L).

11. To obtain a pure virus stocks, choose a clone from a single well on a plate that

yielded the fewest number of positive wells scored by both means ( see Note 14 ).

12. Amplify the recombinant baculovirus clone of choice by repeating step 8 , using

the remainder of the medium in the well as inoculum. Be careful to note increas-
ing passage numbers throughout ( see Note 15 ). Monitoring of the recombinant
protein by Western blotting is recommended after each passage.

13. Prepare a viral stock that will be used for future manipulations and determine the

actual viral titer by end point dilution. Typically, viral stocks of 500 mL are
sufficient for most purposes, and should be stored at 4 C. Shelf-life of viral stocks

should be considerable, with titers being recalculated upon prolonged storage.

°

3.3. Generation and Purification of Recombinant G Domain
14. 10 6 cells/mL.

The culture volume should not exceed 600 mL/spinner flask ( see Note 7 ).

Expand Sf9 cells in 3 1 L spinner flasks to a cell density of 1 - 2 ×

15. Add the viral stock to each of the flasks such that the ratio of recombinant

baculovirus:Sf9 cells is 5:1. Allow the virus to adsorb 1 h, undisturbed, at room
temperature.

16. Incubate 72 h at 27 ° C while stirring at approx 100 rpm.

17. Pellet the cells by centrifugation (875 g , 15 min, 4 ° C) ( see Note 16 ). Transfer the

medium to a prechilled flask and keep on ice.