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From 2013.igem.org

Introduction

To distinguish extracellular proteins from cytoplasmic proteins, extracellular proteins are provided with a cleavable signal peptide. In general proteins are translocated in an unfolded state through a channel composed of integral membrane proteins. The precursor proteins are synthesized at the ribosome and are prevented from tight folding or aggregation, by chaperones. At this stage to possible pathways can be followed. The ribosome can synthesize the precursor protein directly at the translocation channel, called co-translation translocation. The other possibility is to synthesize the precursor protein prior to the translocation (post-translational translocation). After the translocation of the precursor protein, the signal peptide is cleaved off by signal peptidases during or shortly after the translocation [van Wely et al. 2001].

In our design we are employing the Gram-positive model bacterium Bacillus subtilis to secrete spider silk. B. subtilis is generally regarded as safe (GRAS) and is often used in industry for the commercial production of extracellular proteins because they only need to traverse a single membrane. In order to secrete the spider silk protein by the Sec-system of B. subtilis, we constructed various signal peptites in front of our silk protein that will be recognized by the signal recognition particle (SRP) and will be traversed over the membrane.

B. subtilis protein translocation machinery.

Figure 1. Schematic overview of the B. subtilis protein translocation machinery. The signal recognition particle (SRP)
consists of scRNA, Ffh and HBsu. See text for details. The dashed line arrow indicates an SRP-independent pathway,
possibly mediated by SecA that shuttles between the SecYEG-bound and free cytosolic state. Proteases that degrade
the secreted proteins are located near the membrane surface, in the cell wall and free in the suspending medium.

KH van Wely et al. 2006 (FEMS Micro. Rev.)

MotB

FliZ

EstA

LytB