Dear Dipankar, This is a common problem. If you say you have expression, then I would say its not a promoter issue (I usually use polyhedrin). In our lab we use always the native signal peptide, azuricidin, lobster hemolin amongst others, until we find one that works. We also try to use predictions to see if perhaps the length between the signal peptide and the N-terminus is correct (if you are using a non-full length construct, and so your truncation might be too short, and thus interfere with secretion.
Best Kelvin -- Kelvin Lau Structural Plant Biology Laboratory Department of Botany and Plant Biology Science III University of Geneva 30 Quai E. Ansermet 1211 Geneva Switzerland Email: [email protected] <mailto:[email protected]> Phone: +41 22 379 3026 > On May 15, 2017, at 10:44 , Dipankar Manna <[email protected]> wrote: > > Dear Community, > > My apology for the off-topic question! > > I am trying to express a 65 kDa human protease in insect cells using > baculovirus expression system (Invitrogen). I used HBM secretion signal > sequence to expression my protein of interest. Virus generation and titer > amplification was done in Sf9 cells, and the protein was expressed in Hi5 > cells. The western blot shows the expression of protein in the cells, but not > in the supernatant, that means the protein is not secreted (I fear). Though > it should be secreted in the medium as the HBM secretion signal sequence > (Invitrogen) was there at the N-terminus. Also, my protein is a secreted > protein, but I replaced the native signal peptide with the HBM signal > sequence fearing that the mammalian signal peptide might not work in insect > cell (though I am not sure)? If anybody has experienced the same, please > share your experience and suggest me the further steps. Is it possible that > the native signal is necessary for the secretion, even in the insect cell > expression? Also what other signal sequences could be used instead of HBM? > > Thanks in advance for your suggestions/comments! > > Best, > > Dipankar > > -- > Dipankar Manna, Ph.D > Postdoctoral Researcher > Department of Molecular Medicine > Institute of Basic Medical Sciences > University of Oslo, Domus Medica > Oslo, Norway > > Mob : +47 451 66 517 > E-mail: [email protected] <mailto:[email protected]> > [email protected] <mailto:[email protected]> > http://www.med.uio.no/imb/english/people/aca/dipankam/ > <http://www.med.uio.no/imb/english/people/aca/dipankam/>
