Variable Expression of Multi-Component Protein Complexes

Variable expression of multi-component protein complexes is increasingly necessary as more sophisticated projects are attempted.  But how can this be achieved?  Most of our expression work is based on the baculovirus system, in particular our flashBAC™ vectors that permit easy recombinant virus generation in a one-step process.  These insect-specific viruses offer an ideal platform for producing a wide range of recombinant proteins including nuclear, cytoplasmic, membrane-bound and secreted examples. But

Intermediate Scale Recombinant Protein Production

Between initial tests and larger scale up you may want to conduct intermediate scale recombinant protein production using baculovirus vectors.  If you are working with several constructs then this can begin to get expensive of cell culture vessels such as the ubiquitous 125ml conical flasks, in which you can amplify 25-30mls of virus-infected cells.  Space on shakers can also be at a premium in some labs. To address both the

Adapting Insect Cells to a New Growth Medium?

Have you ever had the experience of adapting insect cells to a new growth medium?  We recently had to do this for a client project because they had used a particular medium previously and were keen to maintain the same conditions to continue the work with us. This process made us realise that switching to a new serum-free insect cell culture medium can result in short term stress responses in

Baculovirus-Expressed Secreted Recombinant Proteins

A question that often comes up from clients is what is the best signal peptide to use for baculovirus-expressed secreted recombinant proteins?  While we have discussed the best flashBAC™ variant to use for the production of secreted proteins in a previous blog, we have never considered the role of the signal peptide. In fact, while writing this blog, we realised that there was much that we didn’t know, so will

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. 

Healthy Culture of Insect Cells

Following on from our last blog on the revival of cells stored in liquid nitrogen, we now provide some tips on the continued healthy culture of insect cells.  Whether you are maintaining cells as monolayer or suspension (shake or spinner) culture you will probably faithfully record your name/medium used/date of sub culture/passage number on the side of your new flask.  Alternatively, instead of using a new flask you may simply

Cryogenic Storage and Revival of Insect Cells

Cryogenic storage of cells is essential to ensure long term, convenient access to new stocks. How well this process is conducted can have a significant impact on the revival process too. In this article we provide a few tips on how both freezing and revival of insect cells can be successfully accomplished.

Insoluble Protein – Inconsolable?

You may have had the experience!  An expression project is going really well.  You inserted your gene into a baculovirus vector (preferably one of our flashBAC™ range), amplified the virus, tested expression, obtained a rewarding blob of stained protein on a gel, went on to purify it – only to find an insoluble protein in your hands.  Cue to retire to the bar for a consolation beer – in moderation

Protein Expression and Directed Mutations

If you want to make a modified form of a protein as a recombinant product, this can have unintended consequences on its synthesis in your chosen expression system. Even minor changes to a sequence can affect structure, rendering it difficult to produce or unstable with the result that protein yields are severely reduced. Avoiding this problem may be difficult, particularly if you want to study a particular domain of a protein via mutagenesis. However, including appropriate controls, such as an unmodified version of the protein can be very useful in troubleshooting these problems. Even changes to gene codon usage, while not affecting protein sequence, may affect secondary mRNA structure and thus reduce the translation.

P10 – the Forgotten Baculovirus Hyperexpressed Protein

The ubiquitous polyhedrin gene promoter, from which most baculovirus expression vectors are developed, has an oft forgotten rival – the p10 gene promoter.  The baculovirus expression system, on which our flashBAC™ vectors are based, derives from the original strategy whereby the virus polyhedrin gene coding region is replaced by the target recombinant gene sequences.  These sequences remain under the control of the polyhedrin gene promoter to obtain high levels of

Louis Pasteur and Insect Viruses

We told you last time about our trip to the Society for Invertebrate Pathology meeting in Tours, France.  This was another highly enjoyable gathering.  It also proved to be very successful for two of our OET-sponsored PhD students at Oxford Brookes University.  Mine Aksular won second prize for her poster presentation, “Improving baculovirus surface display system”. Leo Graves had a commendation for his talk, “3-Dimensional ultrastructural modelling of Autographa californica

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