The PelB signal sequence is a small, specialized segment found at the beginning of certain proteins. It acts like an internal address label, directing the protein to a specific destination, typically outside the bacterial cell’s main compartment. This sequence originates from the pectate lyase B gene, initially identified in the bacterium Erwinia carotovora. Its primary function is to guide newly synthesized proteins for secretion.
How Proteins Leave Bacterial Cells
Bacterial cells possess machinery to transport proteins beyond their internal cellular environment. One common route is the Sec (Secretion) pathway. This pathway begins with chaperones, which bind to newly synthesized proteins, keeping them in an unfolded state. This unfolded conformation is important for allowing the protein to fit through narrow channels.
The unfolded protein then associates with the Sec translocase complex, a channel-forming assembly embedded within the bacterial inner membrane. This complex facilitates protein movement across the membrane. Energy for this transport is often supplied by adenosine triphosphate (ATP) hydrolysis, which drives conformational changes in the Sec machinery. A signal sequence, like PelB, is a short amino acid stretch at the protein’s N-terminus. This sequence serves as a recognition tag, directing the protein to the Sec translocase. Once the protein has navigated through the channel and reached the other side of the inner membrane, the signal sequence is typically removed, allowing the mature protein to fold into its active shape.
The PelB Signal Sequence and Its Role
The PelB signal sequence is a distinct N-terminal leader peptide, typically around 22 amino acids. Its structure has three discernible regions that facilitate protein secretion. The N-region, at the beginning, usually contains one or more positively charged amino acids. These positive charges interact with negatively charged components of the bacterial membrane or the Sec translocase, initiating the targeting process.
Following the N-region is the hydrophobic H-region, consisting of 7 to 15 nonpolar amino acids. This hydrophobic segment inserts into and spans the lipid bilayer of the bacterial inner membrane. Its interaction with the membrane forms a temporary pore or channel, through which the rest of the protein can pass. This hydrophobic core is a defining feature of signal sequences that utilize the Sec pathway.
The final part is the C-region, a polar segment that includes a specific cleavage site recognized by signal peptidase. Once the protein has translocated through the inner membrane, Type I signal peptidase cleaves the PelB sequence. This removal releases the mature protein into the periplasmic space, the region between the inner and outer bacterial membranes, or directly into the extracellular environment. The PelB signal sequence thus acts as a temporary guide, directing proteins from the cytoplasm to an external location.
Using PelB in Biotechnology
The PelB signal sequence has become a valuable tool in biotechnology, particularly for recombinant protein production. Scientists attach the gene encoding the PelB sequence to the gene of a desired protein. When this modified gene is expressed in bacteria, notably Escherichia coli, the PelB sequence directs the newly synthesized fusion protein for secretion into the periplasmic space.
Directing proteins to the periplasm offers several significant advantages. The cytoplasm of E. coli is a highly reducing environment, which hinders the formation of disulfide bonds often necessary for the correct folding and stability of many eukaryotic proteins. The periplasm, in contrast, is an oxidizing environment that naturally supports the proper formation of these disulfide bonds. This environment aids in achieving the correct three-dimensional structure for complex proteins.
The periplasm is also a less crowded environment compared to the cytoplasm, which can reduce the likelihood of protein aggregation and improve overall folding efficiency. Isolating proteins from the periplasm is often simpler than purifying them from the cytoplasm. Enzymes, antibody fragments, and various therapeutic proteins are examples of molecules successfully produced using the PelB signal sequence to achieve periplasmic localization.