Part:BBa_K1909003

EnvZ Signal Peptide and first Transmembrane Domain

BBa_K1909003 is a protein head domain part for design of EnvZ chimeric receptors. EnvZ is part of the well-studied and commonly used two-component system EnvZ/OmpR in Escherichia coli, offering a simple way of signal transduction.

Usage and Biology

It was shown that the periplasmic domain of osmolarity sensor protein EnvZ can be exchanged by ligand binding domains of other receptors without compromising its activity (ref. iGEM Tokyo 2012). For easy fusion protein design, we split EnvZ into two parts, removing the periplasmic ligand binding domain to be replaced by other ligand binding domains of interest. This part is designed to form the head domain of EnvZ chimeric receptors, consisting of the n-terminal EnvZ nonstandard signal-peptide like sequence inducing membrane localization, a FLAG-tag for validation of correct expression and membrane orientation as well as the first transmembrane domain.

Functional Parameters

To experimentally confirm the expression of EnvZ-LRR we added a FLAG tag to the sequence. To not interfere with any important protein structure or function, we analysed possible locations for the FLAG tag. EnvZ features a non-standard signal peptide-like sequence at its N-terminus predicted to play an important role in membrane incorporation and topology that is cleaved off posttranslationally. We used the ProtScale algorithm using the hydropathicity scale as described by Kyte and Doolittle to predict the position of the first transmembrane helix of EnvZ (Fig. 2). The first transmembrane helix was predicted to start at L16 of EnvZ. Thus, we inserted the FLAG tag downstream of the predicted signal peptide and upstream of the first transmembrane helix of EnvZ, shifting the first transmembrane helix downstream by 9 residues.

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]

chassis

E. coli JW 3367-3