E. coli is often the host strain of first choice for the production of recombinant proteins. Despite its long and successful history, several limitations remain when the desired protein cannot be expressed in a functional state. This is often due to the inherent properties of expressingheterologous proteins in the cytoplasm. One means of overcoming these impediments is to express the protein such that it is secreted into an environment that is more conducive to correct folding and where proteolytic degradation is minimized, such as the periplasm or extracellular matrix. Periplasmic or extracellular protein export has been exploited for the production of a number of recombinant proteins. The extracellular accumulation of a target protein provides several advantages. These include: A Simplified Downstream Purification Scheme, Enhanced Biological Activity, Higher Product Stability and Solubility.
Protein secretion in E. coli and other Gram-negative bacteria is an extensively reviewed subject.1 It is also a very complex process. The pAES30 series vectors utilize signal sequences that represent the three translocation pathways of the type II secretion process of E. coli.
The type II secretion mechanism is a two-step process. Protein translocation through the inner membrane is accomplished by way of one of three pathways: SecB-dependent (SEC), signal recognition particle (SRP) or twin-arginine translocation (TAT) (Fig. 1). Most proteins in E. coli are exported to the periplasm via one of these pathways with the larger majority using SEC. Extracellular secretion by a type II mechanism is via the main terminal branch pathway, which involves a poorly defi ned complex of 12-16 proteins that are not expressed under normal laboratory conditions.2 Therefore, most of the protein exported by the type II systems remains resident in the periplasm, which is also where most secreted recombinant proteins accumulate.3
Test Principle:Six signal sequences representing the three translocation pathways of E. coli (SRP, SEC, TAT) are fused to the N-terminus of the Target Protein facilitating its secretion to the periplasmic space. Translocation through the inner membrane causes removal of the signal sequence resulting in a Target Protein with its native N-terminus. Once in the periplasmic space, the Target Protein can then be recovered by using any number of reagents that perforate the outer cell membrane, causing release of the Target Protein into the culture medium. Alternatively, cells can be harvested by centrifugation and a cold osmotic shock protocol used to extract the recombinant Target Protein from the periplasmic space.
Storage and Stability:Store powder at 4°C liquid at -20°C. Store other components at 4°C. Stable for at least 6 months For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
