Protein Expression

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? (more…)

Advertisements

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 achieve efficient infection with each recombinant.  But how many viruses can you add to each cell and still get each one into every cell in equal numbers? (more…)

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. (more…)

Insoluble Proteins – 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 and insoluble protein in your hands.  Cue to retire to the bar for a consolation beer – in moderation of course! (more…)

Protein Expression and Directed Mutations

We get asked to do many service projects for customers where they want to produce a modified form of a protein for structural/functional studies.  Frequently, these represent examples that have already been expressed in other studies, often to high levels, so the expectation is that yields will be good in these new projects. Everyone sets off on the road to discovery with high hopes.  Usually, these are fulfilled and both parties are happy! (more…)

Baculovirus Gene Mutations and Protein Expression

Have you ever wondered why the baculovirus we use for most protein expression purposes is called AcMNPV?  This is short for Autographa californica nucleopolyhedrovirus, which derives from the Latin name of the alfalfa looper, a pest of alfalfa crops.  The convention for naming baculoviruses is to use the host insect from which they were isolated.  This does mean that some viruses can effectively be named twice, if they are isolated from different insect species.  (more…)

Producing Secreted Proteins

We had promised to consider the production of virus like particles in this blog, but have postponed this topic to a later date after the subject of optimal secretion of recombinant proteins came up at a recent meeting we attended.  It seems some of you are reporting poor yields of secreted protein using baculovirus vectors after preliminary studies of transient expression in insect cells suggest they should be much higher.  We think we can offer some solutions to this. (more…)

Protein Purification

After scaling up recombinant baculovirus-insect cell cultures (blog) we now come to what can be a very tricky part of the operation, namely how do you extract your protein?  Hopefully, this process was in your mind before you even started cloning your gene of interest into a transfer vector.  For most recombinant proteins it can be convenient to add a string of histidine residues to either end of your target so that you can readily extract it using affinity chromatography.  Other tags can also be used.  You might also consider incorporating a protease cleavage site between the tag and the body of your protein so that you can separate the two sequences in a final purification step.  (more…)

Scaling Up Protein Production

Once you have decided on the optimal conditions for producing your recombinant protein target in baculovirus-infected insect cells, your thoughts may turn to scaling up its production prior to purification.  You may be fortunate enough that the yield of recombinant protein is very high and your requirements fairly modest that only a small volume of virus-infected cells is required.  On the other hand, you may have lofty ambitions for your protein target and you need to produce many litres of starting material prior to extraction and purification.  In this blog, we discuss some simple options for achieving the scale of protein production you need for your particular project. (more…)