Signal peptides for recombinant protein secretion in bacterial expression systems

Abstract

The secretion of biotechnologically or pharmaceutically relevant recombinant proteins into the culture supernatant of a bacterial expression host greatly facilitates their downstream processing and significantly reduces the production costs. The first step during the secretion of a desired target protein into the growth medium is its transport across the cytoplasmic membrane. In bacteria, two major export pathways, the general secretion or Sec pathway and the twin-arginine translocation or Tat pathway, exist for the transport of proteins across the plasma membrane. The routing into one of these alternative protein export systems requires the fusion of a Sec- or Tat-specific signal peptide to the amino-terminal end of the desired target protein. Since signal peptides, besides being required for the targeting to and membrane translocation by the respective protein translocases, also have additional influences on the biosynthesis, the folding kinetics, and the stability of the respective target proteins, it is not possible so far to predict in advance which signal peptide will perform best in the context of a given target protein and a given bacterial expression host. As outlined in this review, the most promising way to find the optimal signal peptide for a desired protein is to screen the largest possible diversity of signal peptides, either generated by signal peptide variation using large signal peptide libraries or, alternatively, by optimization of a given signal peptide using site-directed or random mutagenesis strategies.

Keywords: Gram-positive bacteria; Protein secretion; Recombinant protein production; Sec pathway; Signal peptide; Twin-arginine-translocation (Tat) pathway.

Fig. 1

Two major bacterial export pathways: Sec and Tat. a The general secretion (Sec) protein export pathway. In the cotranslational mode (1), Sec substrates possessing highly hydrophobic signal peptides (SP) are recognized at the ribosome by the signal recognition particle (SRP). Subsequently, the ribosome-nascent chain (RNC)-SRP complex docks to the SRP-receptor FtsY and the RNC is then further transferred to the SecYEG translocation pore such that ribosomal exit site is in close proximity to SecYEG. The energy for translocation in the cotranslational export mode is provided by further elongation of the substrate at the ribosome. In the posttranslational mode (2), Sec-dependent precursor proteins are kept in an export-competent state by posttranslationally interacting proteins (PIP’s) such as SecB, the general chaperones GroELS/DnaK–DnaJ–GrpE or the soluble form of SecA. The signal peptide (SP) is recognized by the SecA protein which pushes the protein through the SecYEG protein conducting channel in a stepwise and ATP-dependent manner. In addition, SecDF exerts a proton motive force (pmf)-dependent pulling force on the substrate from the trans-side of the cytoplasmic membrane (CM). During or shortly after translocation, the signal peptide is removed by signal peptidase (SPase) and the mature protein is released on the trans-side of the CM. b The twin-arginine translocation (Tat) protein export pathway. After folding and, if required (as shown here), cofactor insertion, preproteins containing a signal peptide with a twin-arginine motif (RR) are recognized by a receptor complex consisting of TatC and TatB. Subsequently, homooligomeric complexes of TatA are recruited to the substrate-loaded receptor complex in a proton motive force (pmf)-dependent manner, followed by the translocation of the substrate across the cytoplasmic membrane (CM). Following to substrate translocation, the signal peptide is cleaved by signal peptidase (SPase) and the mature protein is released on the trans-side of the membrane

Fig. 2

General features of Sec and Tat signal peptides. Sec and Tat signal peptides possess a similar tripartite overall structure consisting of a positively charged n-region, a central hydrophobic h-region, and a polar c-region that contains the recognition site (consensus: A-X-A) for signal peptidase (SPase; the cleavage site is indicated by an arrow). In Tat signal peptides, a characteristic amino acid consensus motif including two highly conserved arginine residues (underlined) is present at the boundary between the often significantly longer n-region and the h-region. Furthermore, the h-region of Tat signal peptides is mostly less hydrophobic than those found in Sec signal peptides and in the c-region of Tat signal peptides, frequently positively charged amino acids (the so-called Sec-avoidance motif) are present that prevent a mistargeting of Tat substrates into the Sec pathway

Fig. 3

Signal peptide variation. Influence of 148 different Sec signal peptides from B. subtilis on the secretion of the heterologous target protein cutinase from F. solani pisi into the culture supernatant of B. subtilis. The signal peptides are ranked and numbered according to their performance in cutinase secretion as reported by Brockmeier et al. [58]