Proteomics and protein biochemistry / Protein expression, protein extraction and protein purification / Baculovirus expression system protocols and methods

INSECT CELL CULTURE & BACULOVIRUS INFECTIONS recommended protocol
A detailed protocol on insect cell culture and baculovirus infection. 1.Cells, Media, Growth Characteristics, Freezing and Thawing Cells, and Glassware . 2.Generation of Recombinant Baculovirus. 3.Generation of Recombinant Protein with Baculovirus Infec ...

Sf9 Spinner Culture Media Recipe new protocol
Protocol from Bjokerman group at CIT. ...

Baculovirus Protein Expression Protocol new protocol
Protocol A: Calcium phosphate co-transfection of insect cells Protocol B: Analysis of nonsecreted protein expression Protocol C: Growing and freezing of insect cells Protocols from eBioscience.com ...

Plaque assay for baculovirus culture protocol
There are many ways for virus to hide, and so this assay is usually an underestimate of the actual number of virus present. For instance, a pfu (plaque forming unit) may not find a cell by the time the attachment period is ended or it may stick to the plate wh ...

Freezing and recovering Sf9 Cells protocol
Sf9 cells can be frozen in a manner quite similar to methods used to preserve mammalian cells. This involves putting them in a suitable medium, including 7.5-10% DMSO, and a controlled freezing rate. (Dr. Jim Litts, baculovirus.com) ...

Suspension Culture Infections: Low MOI protocol
Infection of suspension cultures is the most common way of producing protein in the baculovirus system. Suspension cultures are easily scaleable from about 10 mL to more that 100 L. Generally, the suspension cultures are maintained in serum free media. These m ...

Suspension Culture Infections: High MOI protocol
Infection of suspension cultures is the most common way of producing protein in the baculovirus system. Suspension cultures are easily scaleable from about 10 mL to more that 100 L. Generally, the suspension cultures are maintained in serum free media. These m ...

Recovering Virus Stocks from Frozen Cell Pellets protocol
If your recombinant baculovirus stocks have been lost or have "gone off", virus can usually be recovered from frozen pellets of baculovirus-infected insect cells. Virus in these pellets is stable for many years, especially if stored at -70 C or less. (Robin Cl ...

PERSPECTIVES ON BACULOVIRUS EXPRESSION SYSTEMS review
A mini review by Dr. Bart Frank, Arthritis and Immunology Program, Oklahoma state University. In a previous session, advantages of expressing heterologous genes in E. coli were discussed. While high levels of expression in bacterial systems are common, prob ...

Recovering Low-Titer Virus Stocks troubleshooting
Amplifying low titer stock can be a little tricky. The most important determinant of success is the general vigor of the particular construct that you have. Some virus constructs replicate very efficiently, and they're easy. Others are not so cooperative. (Dr. ...

Protein expression in the baculovirus system.
(Curr Protoc Protein Sci. 2001 May;Chapter 5:Unit5.5.)
Bernard A, Payton M, Radford KR.
Glaxo Institute for Molecular Biology, Geneva, Switzerland.

Insect cell-recombinant baculovirus co-cultures offer a protein production system that complements microbial systems by providing recombinant proteins in soluble form and with most post-translational modifications. Moreover, the large size of the viral genome enables cloning of large segments of DNA and consequent expression of complex protein aggregates. This unit describes methods associated with the large-scale production of recombinant proteins in the baculovirus expression system. A method for large-scale production of viral stocks is described and methods for titration of virus are provided (a plaque assay and an end-point assay). Once viral stocks have been prepared and titered, a protocol for testing the virus in small-scale cultures is provided to determine the kinetics of expression, which allows evaluation of various cell culture and infection conditions aimed at developing optimal levels of protein production (e.g., comparisons of different host cell lines, media, and environmental parameters). Support protocols provide instructions for preparing culture samples for protein analysis by SDS-PAGE and discuss analytical methods for monitoring nutrient levels in cell culture fluids. Once optimal process parameters are identified, protocols describe production of the target protein on a large scale in fermentors using either regular batch production in bioreactors or a fed-batch procedure of production in perfusion cultures. Techniques for harvesting cultures from bioreactors are also provided.

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

2: Bouvier M, Menard L, Dennis M, Marullo S.
Expression and recovery of functional G-protein-coupled receptors using
baculovirus expression systems.
Curr Opin Biotechnol. 1998 Oct;9(5):522-7. Review.

3: Roy P, Jones I.
Assembly of macromolecular complexes in bacterial and baculovirus expression
systems.
Curr Opin Struct Biol. 1996 Apr;6(2):157-61. Review.

4: Sorscher EJ, Sommerfelt MA.
Purification of recombinant protein derived from the baculovirus expression
system using glutathione affinity agarose.
Methods Mol Biol. 1995;39:337-48. Review. No abstract available.

5: Potter KN, Li Y, Capra JD.
Antibody production in the baculovirus expression system.
Int Rev Immunol. 1993;10(2-3):103-12. Review.

6: Jackson JA.
Baculovirus expression systems and insect cells.
Bioprocess Technol. 1991;13:402-13. Review. No abstract available.

7: Vlak JM, Keus RJ.
Baculovirus expression vector system for production of viral vaccines.
Adv Biotechnol Processes. 1990;14:91-128. Review. No abstract available.

recombinant bacmid has double bands
(Forum)
baculovirus protein expression troubleshooting

protein induction
(Proteomics forum)
Pet21(d) vector in the E.coli strain BL21 Des (pLys)

Sf9 cells
(Proteomics forum)
Sf9 cells for expression and purification of proteins of human origin in milligram quantities

Technique / Proteomics and protein biochemistry / Protein expression, protein extraction and protein purification / Baculovirus expression system