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
Expression Systems insect cell culture media
OET is pleased to offer a wide range of Expression Systems Insect cell culture media, from the very popular ESF 921, to specialized insect transfection media. Expression Systems’ media are easy to adapt your cells into and the insect specific media gives great protein yield, when compared to rival media. ESF921 insect culture media OET use ESF 921 for all in-house work, due to it’s high performance record, whilst
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
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™
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
Sequence errors in your gene for expression? (Parts 1 + 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
flashBAC 5+1 offer
OET’s flashBAC 5 reaction kit ranges are some of our most popular products. However, recent feedback has revealed that some customers are reluctant to make use of the positive control transfer vector as this sacrifices 1 reaction and so reduces the number of their own viruses that they can produce. In recognition of this issue, until further notice, we are increasing the amount of flashBAC DNA in all of our
Quick guide to plaque assays
Quick guide to plaque assays of baculovirus infectivity. 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
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.