iGEM13_tecMonterrey team has devised a composite part BBa_K1166002, which combined the hlyA-tag, arabinose-inducible hlyB and hlyD as a whole. The hlyA-tag could be fused with the target gene with an internal linker(double GS), and the hlyB and hlyD could be expressed under the induction of arabinose.
Unfortunately, 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.
—The flexible linker BBa_KK3093010 consists of three GS repeats, aiming to join target gene with hlyA-tag while providing larger degree of movement or interaction.
Figure 1. The flexible linker BBa_KK3093010
—The rigid linker BBa_KK3093011 could be translated to AEAAAK-EAAAKA, which could form a helical linkage to keep a fixed distance between the target gene and hlyA-tag and to maintain their independent functions.
Figure 2. The OmpT-cleavable linker BBa_K3093011
—The OmpT-cleavable linker BBa_KK3093012 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.
Figure 3. The OmpT-cleavable linker BBa_K3093012
Figure 4. Plates 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.
Figure 5. Liquid media 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 6. 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. 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 7. 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 green frame, about 26.0kD) clearly surpassed the fusion protein on the gel.
To further demonstrate the mRFP-hlyA was indeed cleaved, we supplemented all the 4 plasmids with his-tag at their N-terminus, which made it possible for us to detect even the marginal expression and secretion of mRFP.
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.
In order to further investigate whether the OmpT-cleavable is functional, and testify the mRFP were expressed in the right size, we add a 6-his tag at the N-terminus of the mRFP-hlyA fusion protein to four types of fusion protein. After using specific testing primer and sequencing to prove the 4 plasmids were correctly constructed, we cultured and amplified the transformants and measure the fluorescence and ran a Western Blot.
Figure 8. WB results of supernatant and pellet of types of linker-transformants.
The WB results indicated that four types of fusion protein were expressed at the correct size and the his-tag was functional after fusion. Unfortunately, we did’t detect a smaller band in the OmpT-cleavable transformant sample, denoting there were no or too little production of hlyA-tag free fusion protein even though we suspected they existed in the SDS-PAGE results.
Figure 9.mRFP fluorescence of supernatant and pellet of types of linker-transformants.
Interestingly, we also noticed that, after the N-ternimal his-tag were successfully added to the fusion protein, the red fluorescence of mRFP decreased dramatically. The positive colonies of four types of transformants were all spotted lighter color compared with negative ones(without his-tag).
Figure 10.mRFP fluorescence variance with different tags
Given this phenomenon, we came up with two possible hypotheses;1). a N-terminal tag has a greater impact on the mRFP’s conformation than a C-terminal tag; 2). the 6his-tag has a greater impact on the mRFP’s conformation than the hlyA-tag.
So we’ve demonstrated that,
- 4 types of tag could be fused with mRFP without changing its fluorescence;
- The tag, to some extent, could indeed affect the fluorescence of mRFP at different terminus.
In conclusion, our 3 types of versatile linker — flexible, rigid, cleavable— were all 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.