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.

Firstly, almost all recombinant proteins are made using coding regions under the control of the very late baculovirus polyhedrin gene promoter.  This gene promoter is active in the very late phase of virus replication, which begins at approximately 18 hours post infection (hpi) and continues to about 72 hpi.  It is the time when the wild type virus is busy occluding virus particles in the nucleus within a polyhedrin protein matrix.  It is not a period when the virus is actively synthesizing high levels of highly processed proteins such as glycoproteins or indeed secreted products.  Most of these are made in the preceding late phase of virus gene expression.

Secondly, beginning at about 8 hpi, the virus starts to make chitinase.  This virus-encoded gene product is necessary for the liquefaction or melting of virus-infected insects in nature.  In the virus-infected cell culture, it serves no useful purpose.  However, owing to a carboxyl terminal KDEL motif, it is retained in the endoplasmic reticulum (ER) of virus-infected cells.  This can be likened to the silting up of a river, which reduces the free flow of water.  The chitinase progressively blocks up the ER, which results in the retention of recombinant proteins in the secretory pathway.  Why doesn’t it prevent virus glycoproteins, such as gp64, the major baculovirus envelope protein component, from being transported to the plasma membrane?  This is probably because the accumulation of chitinase does not become critical until well into the very late phase.

Both of the above factors suggest that the best time to produce secreted proteins in baculovirus-infected cells is during the late phase, between 8-24 hpi.  We were discussing this one day when our CEO woke from his post prandial nap and reminisced about a couple of papers from the ‘90s (we assume the 1990’s and not 1890’s), which tested expression of juvenile hormone esterase1 and urokinase plasminogen activator2 using both late (p6.9) and very late (polyhedrin) gene promoters.  The p6.9, also known as the basic protein, promoter drives expression of a gene product that is required for DNA binding and packaging of the virus genome within nucleocapsids.  In both cases it was shown that more protein was secreted using the p6.9 gene promoter than when the polyhedrin gene promoter was employed.  The study with JHE also showed that better expression was also achieved with the p10 gene promoter than the polyhedrin promoter.  The p10 gene is also expressed in the very late phase, but interestingly its expression begins between 3-6 hours before that of the polyhedrin gene.  This means that significant gene expression is seen in the late phase of expression.  We still don’t fully understand why there is this subtle difference between the expression of the two very late gene products.

Finally, given that chitinase appears to impede progress of secreted proteins through the ER, it also makes sense to use a backbone virus that lacks this gene.  Both flashBAC GOLD and ULTRA lack the chitinase gene and so are ideal for producing either secreted or plasma membrane-bound recombinant proteins.  If you are wondering how to use the late p6.9 gene promoter for recombinant protein production, just purchase either pOET3 or pOET4 from us.  Both vectors incorporate a copy of the p6.9 gene promoter before the same multi cloning site utilised in our popular pOET1 vector.

For more information, click here to visit our OETips section of our website for tutorial, troubleshooting help and more. Otherwise feel free to contact our team who are always happy to help where ever we can, at info@oetltd.com, or visit //www.oetltd.com.

 

  1. Bonning, B.C., Roelvinck, P.W., Vlak, J.M., Possee, R.D. and Hammock, B.D. (1994). Superior expression of juvenile hormone esterase and beta-galactosidase from the basic protein gene promoter of Autographa californica nuclear polyhedrosis virus compared to the p10 and polyhedrin gene promoters.  Journal of General Virology 75, 1551-1556.
  2. Lawrie, A.M., King, L.A. and Ogden, J.E. (1995). High level synthesis and secretion of human urokinase using a late gene promoter of the Autographa californica nuclear polyhedrosis virus.  Journal of Biotechnology 39, 1-8.

CONTACT US

Email: info@oetltd.com

Tel: +44 (0) 1865 483236

Fax: +44 (0) 1865 483236

Mail: Gipsy Lane, Oxford, OX3 0BP, UK.