Biotechnology

Pre-GMP Manufacture of Recombinant Proteins

While OET is currently unable to offer GMP manufacture of recombinant proteins or viruses, we can offer a premium service for pre-GMP manufacture.  This can serve as a very useful bridge to full GMP projects conducted by other service providers.  So what is the advantage of a pre-GMP step? It really boils down to cost.  Some of our own R&D is moving in the direction of needing GMP but we

baculoQUANT™ Titration of Recombinant Baculoviruses

There are many methods available for the titration of recombinant baculoviruses.  One of the most convenient is the use of the quantitative polymerase chain reaction (QPCR), which assesses how much virus DNA is present in a freshly-amplified stock.  This is compared with a standard curve derived from a control virus stock to give an infectious titre expressed as Q plaque forming units (pfu) per millilitre.  OET Ltd market the baculoQUANT™

Co-infection with Baculovirus Vectors for Multiple Protein Expression

A question that comes up from time to time is the use of co-infection with baculovirus vectors for multiple protein expression in insect cells.  The need arises if you have produced a number of baculovirus vectors that each synthesise a component of a multi-subunit complex, such as an enzyme or a virus like particle.  Intuitively, you know that it is probably necessary to add many virus particles per cell to

Sequence Errors in Your Gene for Expression? (Parts I + II)

If you don’t achieve the levels of protein expression expected in your project, have you ever considered that there may be sequence errors in your gene?  This applies to all expression systems, not just those based on baculoviruses.  We should be clear that we are not talking about sequence errors in your gene after it has been synthesized by an out sourcing company.  Quality control by such companies is such

Quick Guide to Plaque Assays

Last week we ran a short training course on baculovirus expression vectors.  As part of this we put together a one page guide for carrying out plaque assays.  This might be useful to those of you looking for something you can have on the lab bench while conducting your own plaque assays.  We have added a image of one of our 12-well plate-based plaque assays below as an example.  The

Transduction of Whole Porcine Kidneys with BacMAMs

One of the unexpected offshoots of the baculovirus expression system has been its development as a vehicle for the delivery of genes to mammalian cells.  These vectors are otherwise known as BacMams.  Although baculoviruses are unable to replicate in mammalian cells, they can enter them via receptor-mediated endocytosis.  In Frederick Boyce’s original paper the Rous sarcoma virus promoter was used to express beta-galactosidase in the HepG2 human liver cell line. 

Producing Virus-Like Particles (VLPs) in Insect Cells

Using baculovirus expression vectors to produce virus-like particles (VLPs) is not new.  Intact, non-infectious poliovirus1 and bluetongue virus2 VLPs were synthesized using some of the earliest baculovirus vectors with considerable success.  In many of the projects we undertake for customers we have also generated VLPs for a wide range of different viruses.  They are ideal candidates for use as sub unit vaccines.  The immunogenic parts of the virus can be

Longevity of Infectious Budded Baculovirus Stocks

A question that comes up repeatedly is the long term stability of recombinant budded baculovirus stocks.  This is a particular issue now that most of them are generated using insect cells grown in serum-free medium.  It has long been a mantra in virology that you should always have a little bit of protein in your virus suspension to help stabilise the virion structure or prevent non-specific binding to glass or

Rapid Virus Titration by QPCR

Knowing the infectious titre of a recombinant virus stock is really important prior to testing for protein production in insect cells.  It enables you to add the optimal quantity of virus to initiate virus infection.  Too much virus and you are wasting your stock.  Too little virus and you won’t establish a synchronous infection.  For some proteins this may not matter, as the virus produced by the initial round of

Virus Like Particles VLPs for Therapeutics and Research

Oxford Expression Technologies (OET) is now able to apply its flashBAC™ baculovirus expression system to vaccine production, particularly in the exciting new field of virus like particle (VLP) vaccines. A virus like particle is an assembly of virus structural proteins that mimics the configuration of a real virus, except that it contains no genetic material. If a person is vaccinated with VLPs then an immune response is generated as if

Titrating Stocks of Recombinant Baculoviruses using the Plaque Assay Technique

We were going to devote this blog to a comprehensive account of how you might determine the infectious titre of your virus stock.  However, when our CEO got wind of this he started ruminating about how things were “back in the day” and how he had to collect seaweed from the seashore to extract agar for his overlays…!  No, we didn’t believe him either, but he is approaching his 7th

Production of High Titre Virus Stocks

In our last blog post we talked about the process of transfecting insect cells with virus DNA and how you could recognize success or otherwise.  Transfections are normally done on a fairly modest scale (2-5ml) in culture dishes.  Therefore your P0 virus stock is of low volume and probably modest infectious titre (107 plaque forming units [pfu] per ml or lower; we will discuss how to measure this value in

Transfection of Insect Cells with Baculovirus DNA

Independent of the method you use to produce recombinant viruses, at some point you have to transfect insect cells with virus DNA to reconstitute infectious virus particles.  When using the flashBAC™ system, you simply add it to your transfer vector containing the foreign gene and use a suitable transfection agent (e.g. baculoFECTIN II )to achieve entry of DNA into the cells.  The transfected cells are then incubated at 28°C for

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