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 (Su, et al., 2012). 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 (Gentschev, et al., 2002).
This is a device that allows a protein to be secreted by means of the alpha-hemolysin secretion system in E. coli. It’s designed so that the only thing that you have to do is to assemble your protein part in the device via the biofusion standard (BBF RFC 23), (Cut the device with EcoRI and XbaI, cut your protein part with EcoRI and SpeI, mix & ligate). This procedure will leave your part in frame with a signal peptide.
Note: It’s important that your part doesn’t contain a stop codon nor a terminator since your protein will be fused at the C-terminus.
Results: Secretion of proteins
Characterization of the secretion complex with GFP
Our initial approach to study the secretion complex was with the use of double Escherichia coli BL21 (DE3) transformants expressing the secretion proteins and two types of GFP: one with a C-terminal HlyA signal peptide and other one with a GFP alone. By comparing the fluorescent signal in the growth media against that one in the soluble fraction of the cell lysates, we expected to quantify the efficiency of the secretion system in translocating a model protein with and without the secretion peptide. Unfortunately, due to the fact that GFP-HlyA was not emmiting any detectable fluorescent signal and that the growth media was interfering with the readings, we were unable to compare the concentration of fluorescent proteins being secreted in each culture.
Characterization of the secretion complex with the therapeutic proteins
Because of the problems enlisted above, we opted for the characterization of this module by using TAT-APOPTIN, TAT-APOPTIN-HlyA, TRAIL and TRAIL-HlyA as model proteins for the secretion system. Besides, thanks to the fact that all of these proteins were HIS6x tagged, we were able to detect their presence by Western Blot using a anti-HIS6x antibody HRP conjugated.
Figure 1: Western Blot, probed with anti-His6x antibody HRP conjugated. Protein samples were recovered from growth media fractions of lysates from E.coli BL21 (DE3) co-transformed with the secretion complex, and purified using HisPur Ni-NTA Purification Kit (Thermo Scientific). Lane1: Purified HIS-TAT-APOPTIN-HlyA (from transformant 2); Lane2: Amersham High-Range Molecular Weight Marker; Lane3: Purified HIS-TAT-APOPTIN-HlyA (from transformant 1); Lane4: Purified HIS-TAT-APOPTIN (from transformant 2); Lane5: Purified HIS-TAT-APOPTIN (from transformant 1); Lane6: Purified HIS-TRAIL-HlyA (from transformant 2); Lane7: Purified HIS-TRAIL-HlyA (from transformant 1); Lane8: Purified HIS-TRAIL (from transformant 2); Lane9: Purified HIS-TRAIL (from transformant 1); Lane10: Positive control (previously confirmed HIS-GFP)
Figure 1 shows the assay for the secreted proteins (revealed with chemiluminescent signal) in the growth media of induced cultures of co-transformed E.coli BL21 (DE3). It can be observed that only two bands are present in the membrane: TAT-APOPTIN (from transformant 2) and TRAIL-HlyA (from transformant 1).
In Figure 2, we show the assay where we tested the expression of therapeutic proteins in the soluble fraction of the lysates; this way, we could detect which proteins are being expressed inside the cells but are not translocated to the growth media.
Figure 2: Western Blot, probed with anti-His6x antibody HRP conjugated. Protein samples were recovered from soluble fractions of lysates from E.coli BL21 (DE3) co-transformed with the secretion complex, and purified using HisPur Ni-NTA Purification Kit (Thermo Scientific). Lane1: Purified HIS-TRAIL-HlyA (from transformant 2); Lane2: Amersham High-Range Molecular Weight Marker; Lane3: Purified HIS-TRAIL-HlyA (from transformant 1); Lane4: Purified HIS-TRAIL (from transformant 2); Lane5: Purified HIS-TRAIL (from transformant 1); Lane6: Positive control (previously confirmed HIS-GFP); Lane7: Purified HIS-TAT-APOPTIN-HlyA (from transformant 2); Lane8: Purified HIS-TAT-APOPTIN-HlyA (from transformant 1); Lane9: Purified HIS-TAT-APOPTIN (from transformant 2); Lane10: Purified HIS-TAT-APOPTIN (from transformant 1)
Although the signal is weak for some lanes, all therapeutic proteins (with the exception of TRAIL from transformant 2) were expressed inside the bacteria. This finding reveals that the HlyA peptide is not enough for the secretion of TAT-APOPTIN-HlyA; but on the other hand, APOPTIN is being secreted to the media even though it has no secretion peptide.
Negative control assay for the secretion of therapeutic proteins
As it has been shown, our previous results indicate that the secretion complex was indeed able to provide a protein secretion mechanism for E. coli nevertheless the presence of TAT-APOPTIN (without the HlyA signal peptide) in the growth medium fraction may suggest three things: a) TAT-APOPTIN is being secreted on its own by an unkwown mechanism, b) the secretion complex may be inespecific in the translocation of some proteins, even when they lack a secretion peptide, or c) The cells are prematurely being lysed before the medium fraction is recovered.
To approach this problem, we induced E.coli BL21(DE3) strains transformed with the therapeutic protein plasmids alone (without the secretion complex) under the same growth and harvesting conditions as the double transformants. By Western Blot analysis, we compared the soluble fractions of the lysate and the medium of the culture (Figure 3).
Figure 3: Western Blot, probed with anti-His6x antibody HRP conjugated. Protein samples were recovered from either soluble or growth media fractions of lysates from E.coli BL21 co-transformed with the secretion complex, and purified using HisPur Ni-NTA Purification Kit (Thermo Scientific). Lane1: Positive control (previously confirmed HIS-GFP); Lane2: Purified HIS-TRAIL (from soluble fraction); Lane3: Purified HIS-TRAIL-HlyA (from soluble fraction); Lane4: Purified HIS-TAT-APOPTIN (from soluble fraction); Lane5: Purified HIS-TAT-APOPTIN-HlyA (from soluble fraction); Lane6: HIS-TRAIL (from medium); Lane7: Amersham High-Range Molecular Weight Marker; Lane8: HIS-TRAIL-HlyA (from medium); Lane9: HIS-TAT-APOPTIN (from medium); Lane10: HIS-TAT-APOPTIN-HlyA (from medium)
The assay reveals that no therapeutic proteins were detected in the growth media, on the other hand though, they were found in the soluble lysate in varying concentrations and as polymers of different sizes. This finding implies that the secretion complex is efectively required for the active secretion of these proteins; even more, as the growth and harvesting conditions were the same, the presence of secreted proteins in the growth media is unlikely to be attributed to premature cell lysis.
In conclusion, the secretion complex was found to be necessary and sufficient in some cases for the secretion of our therapeutic proteins. Further studies would be needed to characterize the efficiency of the secretion of proteins, as well as the properties of the proteins secreted without a signal peptide (like TAT-APOPTIN).
Su L, Chen S, Yi L, Woodard RW, Chen J, Wu J. (2012). Extracellular overexpression of recombinant Thermobifida fusca cutinase by alpha-hemolysin secretion system in E. coli BL21(DE3). Microb Cell Fact. 11:8
Gentschev I, Dietrich G, Goebel W. (2002). The E. coli alpha-hemolysin secretion system and its use in vaccine development. Trends Microbiol. 10(1):39-45.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12Illegal NheI site found at 1550
- 21Illegal BamHI site found at 1489
- 23COMPATIBLE WITH RFC
- 25Illegal AgeI site found at 1324
- 1000Illegal SapI site found at 1306
ECUST-China 2019's improvement
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.
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 retain a certain degree of flexibility.
Figure1:clones 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 an 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 showed that, the fusion protein—mRFP-hlyA(in the red frame, about 32.6kD)—were all expressed inside the cell and partially 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 was because, the pI of the mRFP-hlyA and mRFP were estimated to be 5.12 and 5.07 using an online tool(ExPASy), both lower than pH6.8, which might potentially affect the mobility ratio of these protein in the SDS-PAGE buffer and appeared on a lower molecular weight band 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.
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.
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).
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 the our improved part, BBa_K3093010, HlyA-tag+Secretion system (OmpT-cleavable linker BBa_K3093011) to be functional, and the Rigid linker BBa_K3093012 and the Flexible linker BBa_K3093013 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!
NAU-CHINA 2019's characterization
We constructed the hlyA-rfp plasmid (Fig.1.) and removed the inducible promoter and the double terminator in the HlyA secretion system to construct the simplified hlyA-rfp plasmid (Fig.2.). Then, we characterized the secretion effects of the two systems.
Fig.1. hlyA-rfp plasmid
Fig.2. simplified hlyA-rfp plasmid
In pre-experiment, we found the optimal induction concentration of the arabinose promoter is 0.2% in the unsimplified HlyA system.
32 × 1.2mL LB liquid medium, with 0.2% arabinose and chloramphenicol 50μg/ml.
32 x 1.2mL LB liquid medium, with chloramphenicol 50μg/ml.
32 × 1.2mL LB liquid medium, with 0.2% arabinose and chloramphenicol 50μg/ml.
32 × 1.2mL LB liquid medium, with chloramphenicol 50μg/ml.
Every 1h, a set of samples were placed in the shaker until all samples were placed. The OD600 and fluorescence value of bacterial fluid, the supernatant after centrifugation, and the bacterial liquid resuspended in 1×RBS after removing the supernatant were measured.
The results showed that the HlyA secretion system and the simplified HlyA secretion system had no obvious extracellular secretion effects under 37℃ and 28℃.
To improve the stability of mRNA and ribosome binding probability, we inserted a sequence at the upstream of the RFP coding sequence of the HIyA-RFP plasmid (Fig.3.). Then, we induced the DH5α strain with 0.2% L-arabinose. The bacterial fluids, the supernatant after centrifugation, and the bacterial liquid resuspended in 1×RBS after removing the supernatant were taken, and the OD600 and fluorescence values were measured (Fig.6.).
Fig.3. HIyA-RFP plasmid with UTR inserted.
The results showed that the re-improved HlyA secretion system also did not meet the expected extracellular secretion effect (Fig.5.). But the expression of RFP was improved greatly.
Fig.4. Fluorescence intensity in the supernatant.
Note: The negative values shown in this figure are caused by the error of the background, but it does not affect the relations of the fluorescence values at different times of each treatment.
Fig.5. Fluorescence intensity in the bacterial fluids.
Note: This figure shows the total fluorescence intensity of the bacterial fluids.
Fig.6. OD600 of E. coli, resuspended with 1xPBS 320μL.
Note: -ara: no 0.2% arabinose induction
+ara: induction culture with 0.2% arabinose for 16 hours
-utr: no BBa K3139001 insertion
+utr: BBa K3139001 insertion
We verified the BBa_K1166002 again, finding the secretion effect is not significant.
However, the secretion effect decreased after L-arabinose induction. And the total amount of fluorescence also decreased with the use of L-arabinose. At the same time, we measured that the numbers of bacteria in different treatments, which were at the same level (Fig.6.).
After two experiments, we believe that the function of BBa_K1166002 HlyA secretion system cannot be observed under our experimental conditions. We have proposed the following possibilities and these may be verified in future research.
1.How to explain the abnormal function of BBa_K1166002 HlyA secretion system?
According to some existing reports, the transmembrane complex of the secretory system also has a TolC protein, but its coding gene is not presented in the part. So, the function of the transmembrane complex that is encoded by the part may be missing, and the RFP they express will be retained in the intracellular and periplasmic spaces. Therefore, we cannot observe the realization of its function.
2.How to explain the extracellular fluorescence?
Since the number of bacteria is kept at the same level, we can roughly rule out cell lysis due to protein accumulation. We hypothesized that there is another mode of transportation in E. coli DH5α that can transport excessive fluorescent protein to the extracellular domain, and this transport mechanism will work faster with the increase in the number of fluorescent proteins.
3.Why did the L-arabinose induction reduce the total fluorescence?
L-arabinose induced the expression of hlyB and hlyD in BBa_K1166002, which are functionless, and even a waste of cell’s energy, since the effective transmembrane complex is not available. So the resources that should be given to the expression of rfp reduce, leading the decrease of its expression and fluorescece intensity.
4.How to explain the phenomenon described in 2 is more obvious after inserting UTR?
The expression product of BBa_K3139001 UTR can raise the stability of mRNA and promote the combination of ribosomes and mRNA. So there is more rfp expression. Besides, without hlyB and hlyD’s competition, more resources can be given to rfp expression.
Functional Parameters: Austin_UTexas
Burden Imposed by this Part:
Burden is the percent reduction in the growth rate of E. coli cells transformed with a plasmid containing this BioBrick (± values are 95% confidence limits). This BioBrick did not exhibit a burden that was significantly greater than zero (i.e., it appears to have little to no impact on growth). Therefore, users can depend on this part to remain stable for many bacterial cell divisions and in large culture volumes. Refer to any one of the BBa_K3174002 - BBa_K3174007 pages for more information on the methods, an explanation of the sources of burden, and other conclusions from a large-scale measurement project conducted by the 2019 Austin_UTexas team.
This functional parameter was added by the 2020 Austin_UTexas team.