A general outline for baculovirus expression is listed below. For one project, it usually takes approximately 2 months to accomplish these goals:
-Target gene will be cloned to a bacterial transfer vector with enhancer in polyhedrin promoter. The inserts are typically flanked by portions of viral genes to permit homologous recombination with replication defective, linear, viral DNA. Vectors or inserts may include 6 x his-tag sequences for protein targeting and purification. Direction of the inserts relative to the polyhedrin promoter will be verified and plasmid for co-transfection will be purified.
-Recombinant viruses are formed after intracellular homologous recombination between the ends of the viral molecules and portions of the transfer vector that flank the gene of interest. These recombination events insert your gene into the virus and complement defective viral gene(s) to permit viral replication. Non-recombinant viruses are kept to a minimal within this system.
-Transfected cell supernatants are harvested. Insect cells with dilutions of this supernatant are infected to isolate single virus plaque. Viral plaques in 3 ml infection are identified and can also be detected via Western blots.
-Additional insect cells are infected with viruses from these plaques to amplify the high titer of viral stocks. Protein expression in these cells may be examined in Western blots.
-The level of protein expression (MOI, time course of infection) is optimized and its activity is tested in the appropriate bioassay.
It is common to obtain high protein yield from E. coli expressions system. However, problems with proper folding and lack of post-translational processing may produce nonfunctional recombinant proteins. The Baculovirus Expression Vector System can be used to prevent, improve, and alleviate these problems. Heterologous gene expression in baculovirus systems offers several advantages over E. coli systems. Meanwhile, these viruses are considered safe for laboratory personnel. Insect cells infected with the virus recognize and properly process a variety of recognition signals in mammalian, plant and yeast genes, and provide moderate to high yields of biologically active proteins for study, diagnostic analysis and the other purposes etc. For the past twenty years in baculovirus development, scientists have not only been able to easily and conveniently express target protein in BEVS, but also developed various methods (serum free medium, insect cell lines and baculovirus virus genome etc.) to improve its efficiency.
In today’s world, more scientists are working on baculovirus expression systems for functional recombinant proteins and more people are discovering more complicated procedures to generate recombinant virus and produce recombinant proteins. However, it is very expensive to train technicians and set up a laboratory suitable for baculovirus expression. Thus, outsourcing is an alternative way to produce recombinant protein for current projects. Not only will you save money, but most importantly, you will also save time.
To understand outsourcing of protein project, the following should be considered:
Is it worth outsourcing to the BEVS laboratory?
How much does a project cost?
What is the typical yield of recombinant protein from an one liter expression?
When will the outsourcing project be finished?
Who is working on this project?
Here at Sheatech, Inc., we provide excellent outsourcing Baculovirus Expression Services. Our scientists in the department of R & D in Sheatech, Inc., have discovered several factors, which aid in the improvement of protein expression in Baculovirus Expression System. Not only can high quality baculovirus virus genome DNA generate high titer recombinant virus, but the enhancer in polyhedrin promoter has also provided and has been proven to show an increase in protein expression for most recombinant proteins. Both mid log insect cell culture as well as optimization conditions of infection have been considered to produce successful virus formation and protein recovery.
1) What are the main differences between E. coli expression and Baculovirus expression?
Baculovirus expression is an eukaryotic expression system with post-translational modification during protein expression whereas in E.coli is a prokaryotic expression system without post-translational modifcation. Eukaryotic genes can produce soluble recombinant protein in Baculovirus expression system.
2) How do you grow healthy sf9 cells?
It is imperative that you choose the right medium and incubation conditions for the insect sf9 cells. Make sure you grow the sf9 cells in log phase and low passages free of overgrowth and with the proper amount of dilution. Check to make sure that the monolayer is free of enlarged cells as well as floating cells.
3) What type of medium does insect cells grow in?
Insect cells can grow in serum free medium and/or complete medium (TNM-FH, 10% FCS). It is important to grow sf9 cells in log phase with serum free or complete medium. It usually takes several passages for the insect cells to adapt to the serum free medium. Once insect cells adapt and grow in serum free medium, they can grow well in monolayer culture and suspension culture in a spinner flask.
4) How do you prevent contamination of co-transfection?
Be sure to confirm transfection reagent and medium with your vendor. Make sure that the DNA sample is suspended in sterile TE buffer. Add suitable antibiotic to the medium for co-transfection.
5) Why has co-transfection failed to produce recombinant virus?
Call vendor to make sure that the lot of virus DNA is indeed suitable for co-transfection. Check the condition and density of seeding sf9 cells. If sf9 cells are seeded too densely, then they might generate low titer virus during co-transfection. The virus titer from the co-transfection may be too low to identify the infected sf9 cells. By using virus amplification, the insected sf9 cells can be identified by microscope.
6) Why aren't there any plaques on the plaque assay?
Make sure the sf9 cells are healthy and suitable for plaque assay. Be sure to check the density. Virus titer may be too low or too high to be detected on the plaque assay. In general, four serial dilutions of virus should be enough to cover the broad range of virus titer. Ensure that the temperature of agarose layer for plaque assay is appropriate. Be sure to prepare fresh Neutral red solution for each plaque assay.
7) How do you generate high-titer virus stocks?
Start from a plaque and amplify titer from a small volume to a large volume using sf9 cells. Make sure you infect healthy sf9 cells for high titer virus.
8) How is the stability of the high titer virus?
High titer virus is stable in FCS medium for a couple of months. Virus titer can be determined by plaque assay or end point dilution. Confirm virus titer first and infect healthy sf9 cells for protein expression.
9) How do you prevent cracks on the plaque assay and allow plaques to grow around the center region of the plate?
Be sure to add 1mL of medium once agarose layer has set. The liquid medium prevents cracks on agarose layer during incubation. Seed healthy sf9 cells uniformly in the dish, add diluted virus solution, and gently remove the virus completely; by doing so, the plaques will locate uniformly in the dish.
10) Why is it so important to optimize conditions for protein expression?
Both recombinant virus and recombinant protein might affect protein expression in Baculovirus expression system. A better and higher yield will result under optimized conditions. Both MOI and insect cells lines are used to find out optimized conditions.
11) How much protein will be produced from one-liter insect cell expression?
The amount of protein produced will range from 10mg/liter to 100ug/liter; this depends on the genes and the growing conditions. Most of the proteins are soluble in the Baculovirus expression system; however, some proteins are insoluble in this system. Also, neutral detergent may help the solubility of recombinant proteins.
*Please Note: Sheatech, Inc. has provided the above questions and short answers/suggestions to these common problems. If you are, however, facing an unusual issue or problem not listed above, please feel free to contact Sheatech, Inc. at (619) 788-8188 or email us at info@sheatechinc
His tag and purification