Part:BBa_K157001

EGFR/ErbB1 signal peptide; mediates protein transport to a translocational pore

This sequence mediates transportation of the protein to a translocational pore by an RNA–multiprotein complex, the signal recognition particle (SRP) when fused to the n-terminus of a fusion protein with a transmembrane region[1]. There, after a pause of translation, the signal sequence is released and translation and translocation of the nascent chain are restarted[2].

Additional characterization of BBa_K782063 by [http://2016.igem.org/Team:LMU-TUM_Munich Munich 2016]:

This BioBrick could be identified as working.

Results of signal peptide functional analysis

Bioinformatic signal peptide analysis via the SignalP server

The [http://www.cbs.dtu.dk/services/SignalP SignalP] server, being able to discriminate between the hydrophobic signal peptide sequence and the hydrophobic transmembrane domain, can determine whether a sequence possesses the biophysical requirements for functioning as a signal peptide. Furthermore, it is able to predict the probable cleavage site of the signal peptide after its translocation into the ER. For all constructs, signal peptide functionality is predicted, as well as a potential cleavage site. The complete translated amino acid sequences of the respective receptor constructs were used as input. The algorithm for eukaryotes with default D-cutoff value was chosen.

Figure 2: A) Schematic depiction of a protein-coding mRNA containing a 5'- and 3'-UTR, the first of which being an important factor in translation efficiency. B) Identification of signal peptide functionality and cleavage site within the receptor construct containing the EGFR, Igκ and BM40 signal peptide via the SignalP 4.1 server. Plotted probabilities represent the raw cleavage site “C-score”, the signal peptide “S-score” and the combined cleavage site “Y-score”. C) ClustalW multiple sequence alignment of the BM40, EGFR and Igκ signal peptide construct, depicting a gap for the EGFR-signal peptide construct compared to the newly designed ones, indicating smaller length of the 5'-UTR . For the Igκ and BM40 signal peptide constructs, a T7 promoter spacer as well as a Kozak sequence increase the distance between TATA-box and protein-coding region.

For all three signal peptides, the high S-score indicates high signal peptide functionality. For the Igκ signal peptide, a cleavage site between amino acid 20 and 21 within the signal peptide is predicted. For the BM40 signal peptide, a cleavage site between amino acid 17 and 18 is predicted, and for the EGFR signal peptide, a cleavage site between amino acid 24 and 25 is predicted.

Quantification of signal-peptide mediated translocation via a secretion luciferase assay

For the quantification of signal peptide functionality via a luciferase assay, three constructs were created and tested - each containing one of three signal peptides (the EGFR signal peptide, the Igκ signal peptide or the BM40 signal peptide) and a nanoluciferase as well as a CMV promoter, a Strep-tag II for immunochemical detection and the hGH polyadenylation signal sequence. Not containing a transmembrane domain, the nanoluciferase fusion protein is being translocated into the ER and then secreted into the medium. Using a luciferase assay, one can quantifiy the amount of luminescence - and thus, proportionally, the amount of secreted luciferase - by measuring the conversion of luciferin into visible light and integrating it over a timespan of 5 s. Therefore, medium samples were taken every 12 h after transfection of cells and measured via the Promega NanoGlo® luciferase assay system according to the manufacturer's instructions.

Figure 3: A) Schematic depiction of the genetic constructs used for signal peptide testing via a secretion luciferase assay. B) Results of the luciferase assay, showing luminescence in relative luminescence units (RLU) as a function of time for the previously described three different signal peptide constructs as well as a control construct containing no signal peptide. C) Detection of secreted luciferases in the medium via a Western Blot, using an anti-Strep-tag II antibody as well as an alkaline phosphatase-coupled secondary antibody for detection.

Discussion: The choice of signal peptide was nailed down

As would be expected, the EGFR signal peptide construct showed the lowest level of secretion. Although one might think this could also be attributed to a lower functionality of the signal peptide sequence itself after being translated, the lower secretion level is more likely to stem from lower translation initiation levels. Almost completely missing a 5’ UTR as well as a Kozak sequence, slower translation initiation (being a rate-limiting step) is likely to have lowered the level of translated protein in total. The constructs containing the Igκ and the BM40 signal peptide, on the other hand, contain a longer 5’ UTR as well as the Kozak consensus sequence, and thus result in higher secretion levels. Since the BM40 signal peptide hereby showed the highest secretion levels, it was thus incorporated into the receptor constructs.

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
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 3
  

[1] Walter P, Johnson AE: “Signal sequence recognition and protein targeting to the endoplasmic reticulum membrane.” Annu Rev Cell Biol 1994, 10:87-119
[2] Robert M Stroud, Peter Walter: “Signal sequence recognition and protein targeting”, Current Opinion in Structural Biology 1999, 9:754–759