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? It really boils down to cost.  Some of our own R&D is moving in the direction of needing GMP but we

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

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

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

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.

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

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. 

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

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

Optimising Protein Expression

Once you have finished making your recombinant baculoviruses expression vector you naturally want to maximize the amount of protein made in insect cells.  A point we have emphasized in a previous blog is the importance of determining the infectious titre of your virus stock accurately.  It is also prudent that you produce a stock of virus large enough that you can conduct a series of experiments to optimise protein production