Part:BBa_K5271004

Secretory peptide for Panobody

A secretory signaling peptide that facilitate the transport of Panobody to periplasm space of E.Coli for secretion.

Profile

• Name: Secretory peptide for Panobody
• Base Pairs: 66 bp
• Amino acid: 22 a.a
• Origin: Escherichia coli
• Property: A secretory signaling peptide from Heat-Stable Enterotoxin II (ssSTII) that facilitate the transport of Panobody to periplasm space of E.Coli for secretion.

Usage and Biology

Protein secretion to the periplasm of E. coli is one of the routes to produce heterologous proteins that contain disulfide bonds. However, it is still challenging to use periplasmic secretion to produce complex heterologous proteins such as full-length monoclonal antibodies (mAbs). The secretion of mAbs in E. Coli often results in proteolytic degradation, protein aggregation, formation of inclusion body. Hence, secretory peptides are commonly used to mediate transportation of the protein from the cytoplasm to the periplasm and thereby facilitate the protein secretion. It has been known that the DNA sequence of the secretory peptide overlaps with the translation initiation region (TIR) [McCarthy & Gualerzi, 1990], and changes in the TIR sequence can significantly impact the periplasmic levels and secretion of heterologous proteins [Simmons & Yansura, 1996]. Zhou et al. tested a panel of secretory peptide derived from the Heat-Stable Enterotoxin II (ssSTII), DsbA, maltose binding protein MalE, and alkaline phosphatase PhoA and confirmed that ssSTII has preferentially mediated secretion of heavy chains of mAbs. Our team added this secretory peptide in front of the coding sequence of nanobody containing the Fc fragment in attempt to solve the problem of insolubility and the formation of inclusion body.

Source

The DNA sequence of secretory peptide is obtained partially from the Heat-Stable Enterotoxin II (ssSTII) of E.Coli.

Design Note

The secretory sequence has mild to insignificant effect in facilitating nanobody with humanized Fc fragment. The nanobody containing this secretory peptide failed to folded into soluble form and secrete to the periplasm space.

Reference

• McCarthy, J. E., & Gualerzi, C. (1990). Translational control of prokaryotic gene expression. Trends in Genetics, 6, 78-85.
• Simmons, L. C., & Yansura, D. G. (1996). Translational level is a critical factor for the secretion of heterologous proteins in Escherichia coli. Nature biotechnology, 14(5), 629-634.
• Zhou, Y., Liu, P., Gan, Y., Sandoval, W., Katakam, A. K., Reichelt, M., ... & Reilly, D. (2016). Enhancing full-length antibody production by signal peptide engineering. Microbial Cell Factories, 15, 1-11.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]