Optimisation and exploitation of SRP-mediated secretion: production of human interleukin-3 and epidermal growth factor by bacillus subtilis (VBI.4837)

Bacillus species are attractive hosts for the production of endogenous and heterologous proteins because these gram-positive bacteria have a high capacity to secrete proteins directly into the extracellular medium. Furthermore, Bacilli are apathogenic, free of endotoxins and well-known with respect to fermentation technology. Recently, evidence has been obtained for a new pathway of protein targeting to the cytoplasmic membrane of B. Subtilis and other eubacteria like E. coli. This conserved pathway resembles protein targeting to the eukaryotic endoplasmic reticulum (ER) and involves the signal recognition particle (SRP). In eukaryotes, the SRP mediates co-translational targeting and membrane insertion by interacting with the targeting sequence of nascent secretory proteins in the cytosol and with the SRP receptor in the ER membrane. In B. subtilis, a small SRP and its receptor have been identified based on sequence similarity studies. Recent studies at Genencor Intl. BV show that depletion of these essential SRP route components in vivo has a pronounced effect on the secretion of endogenous and wspecially on heterologous proteins like human interleukin-3 (IL-3) that are targeted by the SRP in their natural host. This suggests that IL-3 uses the SRP for targeting to the B. subtilis cytoplasmic membrane. Co-translational membrane association by the SRP may avoid the exposure of protein segments in the cytosol that have a tendency to fold or aggregate in a non-productive way and thus inhibit translocation.
In collaboration with the biotechnological company Genencor Intl. BV we will analyse in this project at what level secretion of heterologous proteins is blocked in B. subtilis with a compromised SRP route. The study will concentrete on two human proteins of great interest, IL-3 and epidermal growth factor (EGF). The obtained results will help in optimisation of SRP-mediated secretion of IL-3 and EGF by following a number of strategies.
We will study the role of structural features of the mature protein with respect to targeting via the SRP pathway.
We will make recombinant B. subtilis strains with elevated cellular levels of SRP and SRP receptor (Srb) which are normally not very abundant targeting factors. Furthermore, we will investiga both genetically and biochemically the coupling of the SRP to the B. subtilis translocase via the B. subtilis SRP receptro Srb.
The constructs and strains that originate from these studies will be tested for secretion of IL-3 and EGF on fermentor scale under different growth conditions at the Genencor Intl. BV plant. Furthermore, expecially constructed protease-negative strains will be employed.
The observations made with IL-3 and EGF may open the way to cost-effective production of other four helical bundle human growth factors.

Er zijn drie bedrijven en één andere universiteit bij dit project betrokken.

Dr. J. Luirink
Vrije Universiteit Amsterdam
BioCentrum Amsterdam
Moleculaire Microbiologie
De Boelelaan 1087
1081 HV Amsterdam