Part:BBa_K1166002

HlyA-tag+Secretion system

Introduction

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).

Usage

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).

References

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1550
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1489
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1324
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 1306
  

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