Part:BBa_K3093011

HlyA-tag (OmpT-cleavable linker)

The alpha-hemolysin system is one of the best-studied type 1 secretion systems (T1SS) of E. coli. In T1SS the secretion occurs in a single step directly from the cytosol to the extracellular medium. The secretory machinery of the alpha-hemolysin system consists of three proteins: HlyB, an ATP binding cassette; HlyD, a membrane fusion protein; and TolC, an outer membrane protein.The natural substrate HlyA can be secreted because it contains a C-terminal signal and it has been shown that proteins with C-terminally fused HlyA signal sequence can also be recognized by the HlyB-HlyD-TolC translocator.

Usage and Biology

iGEM13_tecMonterrey team has devised a composite part BBa_K1166002, which combined the hlyA-tag, arabinose-inducible hlyB and hlyD as a whole. Yet the characterization results from iGEM13_tecMonterrey team has shown that, 1).the hlyA-tag could affect the conformation of GFP(the target protein in this case), thus leading to the fluorescence quenching of GFP. This problem implied that, the hlyA-tag might influence the conformation and even function of the target protein when expressed as fusion protein; 2). the hemolysin secretion system is sufficient only to small range of protein. Given these defects existing in part BBa_K1166002, we have improved this part by amending 3 different types of alternative linker—flexible linker, rigid linker, and OmpT-cut linker—on the basis of original double-GS linker.

Characterization

The OmpT-cleavable linker BBa_K3093012 contains an Outer Membrane Protease(OmpT) cleavable sequence — ARRA. We designed this linker, because even though the stable linkage between functional domains could provide many advantages such as a prolonged plasma half-life, nevertheless, they also have several potential drawbacks including steric hindrance between functional domains, decreased bioactivity, and altered metabolism of the protein moieties due to the interference between domains. Under these circumstances, we introduced OmpT cleavable linkers to release free functional domains in vivo. OmpT is a protease localized to the outer membrane and cleaving protein with two consecutive basic amino acids, such as Arg-Arg, Lys-Lys, Arg-Lys. We chose ARRA sequence among the other since it’s the sequence in the standard substrate(Abz-Ala-Arg-Arg-Ala-Dap(dnp)-Gly ) to test OmpT cleavage activity. And the ARRA site is flanked with GS, in order to be exposed to OmpT and flexibility.

Experiment results

Figure1:colonies and of DH5α transformants (pIN2 + mRFP + linker + hlyA + araC + pBAD + hlyB + hlyD) of 4 types of linker on LB-kan plates and in liquid M9 media.

All the colonies proved to be positive by colony PCR and first generation sequencing could be easily spotted red on plates and liquid media, directly indicating that the all the linker+hlyA tags were not interfering the emission of fluorescence of mRFP. Compared with using GFP as the secreted target gene, mRFP is apparently more suitable for the fusion of hlyA to testify the secretion efficiency of hemolysin system.

Figure-2:liquid LB media containing positive colonies with(Y) or without(N) arabinose induction after 16 hours. Left three media are pIN2+mRFP, and pIN2(empty vector) as negative control.

Positive transformants with different linker were cultured and induced by arabinose. It’s conspicuous that the arabinose induced E.coli grew much slower, implying expressing HlyB and HlyD was a addictive burden for the engineered cell. We collected the same weight of E.coli by restricting the value(OD multiple volume) equal to 16.32. And then separate the media and cell by centrifugation. The supernate were concentrated 100 fold and the pellet were resuspended with PBS. Supernate and pellet were both pretreated to prepare protein sample and run SDS-PAGE.

Figure-3:SDS-PAGE of supernatant and pellet of types of linker-transformants.

The SDS-PAGE shows that, the fusion protein—mRFP-hlyA(in the red frame, about 32.6kD)—were all expressed inside the cell and successfully secreted to the media. And in the OmpT-cleavable linker case, the mRFP and hlyA-tag were also successfully cleaved, since the cleaved mRFP(in the rgreen frame, about 26.0kD) clearly surpassed the fusion protein on the gel. Although the mRFP-hlyA fusion protein and mRFP were both slightly smaller than expected size, we highly suspected that’s because, the pI of the mRFP-hlyA and mRFP are estimated to be 5.12 and 5.07, both were lower than pH7, which might dramatically affect the mobility ratio of these protein in the SDS-PAGE buffer and shows at a lower molecular weight on the gel.

So we’ve demonstrated the our improved part, BBa_K3093010, OmpT-cleavable linker to be functional, and the flexible BBa_K3093011 and rigid BBa_K3093012 linker were also proved to be not hindering the secretion of fusion protein while offering several alternative options for perspective users who want to express their protein in an secretory form. We are also looking forward to more application of our secretory system package!

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]