Sequence Errors in Your Gene for Expression Part II?

Sequence errors in your gene for expression Part II follows on from an earlier blog where we discussed a problem we were having producing a particular protein after synthesizing a gene based on very old sequence data. The baculovirus we made containing this synthetic gene produced very low levels of recombinant protein that were hardly visible after immunoblot analysis. This wasn’t a good start to a project where a purified protein was required for diagnostic purposes. (more…)

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Sequence Errors in Your Gene for Expression?

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 that it is extremely rare for there to be a problem.  No, we are referring to data that you may have down loaded from online sources, principally GenBank. These databases host information from the very early days of sequencing, when deriving the sequence of an entire gene was a major achievement. (more…)

OET at ISBioTech Washington 6-8th March

OET founders, Linda King and Bob Possee will be attending ISBioTech in Washington, 6-8th March 2017.  Linda will be presenting a talk entitled, “BacMam Expression Vectors: Applications in Gene Therapy”.  The use of BacMAMs for recombinant gene expression in mammalian is an ever expanding application of baculovirus vectors.  Her talk will encompass some of her research group’s recent work on the potential use of BacMams to transduce whole organs and how this might help in gene therapy to improve the success rate of transplanted organs.  Some of this work was published recently by Hitchman et al.

Sf9 Cells from Monolayers to Suspension Cultures

Transferring Sf9 cells from monolayers to suspension cultures can sometimes be difficult.  These insect cells, commonly employed for baculovirus-mediated expression, are usually kept growing in suspension culture.  However, sometimes monolayer cultures are used to keep them going during periods of low demand.  These cultures are also useful as a reserve or back up for the main suspension cultures, which even in the best laboratories can sometimes become contaminated with unwanted microbial visitors! (more…)

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.  Further work showed that over 70% of cells in primary cultures of rat hepatocytes displayed expression of beta-galactosidase after exposure to the virus.  Although in this study other cell lines did not appear to be able to express the reporter gene, the subsequent use of other promoters, such as that from cytomegalovirus, seems to have overcome this problem.  (more…)

Healthy Cells Over the Holiday

For a follow up to our last blog we want to talk about maintaining healthy cells over the holiday period spanning Christmas and New Year.  Ok, if you are like our CEO you don’t really want to be thinking about the office Christmas party so early, but end of year is creeping up on us.  Most labs, academic and industry, will have some time off. It is often a problem keeping your cell cultures in good condition so that when you return in 2017 you can pick up that important protein expression project without too much delay.  Given that most insect cell lines (usually Spodoptera frugiperda or Trichoplusia ni cell lines) need to be sub cultured at least twice a week if they are grown as suspension/shake cultures, how will they survive for this protracted period? (more…)

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 dilute the cells in their existing vessel and continue to culture them – more about this below.  A question that often vexes people is how many times a culture of cells should be sub cultured or passaged before they are no longer fit to use.  There is no simple answer to this question as it relates to the healthy culture of insect cells. (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…)

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 multicapsid nucleopolyhedrovirus infection in insect cells to determine the role of P10 during baculovirus infection”  He might have received a higher award had the audio visual equipment been able to show properly his movie depicting the 3D model of the P10 protein.  Abstracts for these talks can still be accessed: Mine’s is on page 125 and Leo’s on page 31 of the abstract book. (more…)