Part:BBa_K5136222

I0500-B0034-FhuD-GGG linker-T7 lysozyme 119G-SsrA-B0015

Biology

FhuD

In Escherichia coli, protein translocation guided by signal peptides primarily employs two distinct mechanisms: the Sec- pathway and the Tat- pathway. Notably, some proteins are capable of utilizing both pathways for their translocation (1) . The FhuD signal peptide, acting as an intrinsic dual Sec-Tat pathway (2), is frequently employed in biotechnological applications to direct the secretion of proteins to the extracellular space or the cell membrane. This characteristic makes the FhuD signal peptide an ideal choice for constructing secretion expression vectors, particularly in applications aimed at enhancing the yield of target proteins.

GGG Linker

[(G4S)_n] is commonly used in protein engineering because of its flexibility and resistance to proteases. Therefore, we selected (GGGGS)₃ flexible linker (3) as a short peptide to connect FhuD and T7 lysozyme 119G in our autolytic system.

T7 Lysozyme 119G

T7 lysozyme is a small molecular weight protein in bacteriophage T7, primarily functioning to degrade the cell wall of host bacteria during phage infection, facilitating the injection of phage DNA or the release of newly formed phage particles. In molecular biology research, it is widely used for the efficient lysis of Escherichia coli cells (4, 5). Moreover, it has been reported that higher levels of lysozyme provided by plasmids pLysE or pLysH can reduce the full induction activity of T7 RNA polymerase, allowing induced cells to continue growing indefinitely while producing non-toxic target proteins (5). This feature not only highlights the excellence of T7 lysozyme in promoting cell lysis but also makes it extremely useful in preparing cell extracts for protein purification.

Notably, T7 lysozyme 119G sequence was found in pLysS (6), and it differs from the T7 lysozyme 119V sequence selected from the UniProt database (7), with a variation at the 119^th amino acid position.

SsrA

The SsrA is a small peptide tag used to mark proteins for protein degradation. When fused with the target protein, SsrA could guide it to specific proteases, such as the ClpXP and ClpAP complexes, for degradation (8).

Usage and Design

In our design, we aim to induce cell autolysis to release enzymes into the supernatant, simplifying the complex protein purification process. By utilizing the dual-pathway signal peptide FhuD, we direct T7 lysozyme to the peptidoglycan layer, enhancing cell lysis. Additionally, the SsrA tag is fused to the C-terminus of T7 lysozyme to ensure the degradation of any leaked T7 lysozyme, minimizing system cytotoxicity and ensuring the proper accumulation of the target enzyme in the correct location (9).

This composite part we constructed aims to express the FhuD-T7 lysozyme-SsrA mediated autolytic system (FLSA), which includes T7 lysozyme 119G, under the control of an L-arabinose inducible promoter. To validate the efficiency of the FLSA system, we used sfGFP as a reporter.

Figure 1 The expression gene circuits for the FLSA system.

Characterization

Agarose Gel Electrophoresis (AGE)

The composite part (No part name specified with partinfo tag.