Our project idea focuses on protein purification, a essential technique in synthetic biology. Aqueous two-phase separation (ATPS) is a liquid-liquid fractionation technique effectively used for protein separation and purification. When a protein fuses with a hydrophobin, the hydrophobin changes the hydrophobicity of the protein, which causes the protein to aggregate into the surfactants. Traditional ATPS uses fungal hydrophobins such as HFBI and HFBII. ATPS's industrial application is hampered by the high costs of fungal hydrophobin and the low separation speed (~10 hours/batch). Our team is trying to improve traditional ATPS by incorporating a continuous-flow system and replacing fungal hydrophobins with BslA. We will construct BslA fusion proteins and purify the target proteins using the improved ATPS method. We are confident to achieve a yield higher than traditional ATPS at a faster speed and a lower cost.
Why use BsIA?
Hydrophobins are small surface-active amphiphilic proteins containing both hydrophobic and hydrophilic areas on their surface. They can self-assemble to form a stable film at the interface of both polar and non-polar phases. Hydrophobins have broad application prospects in the fields of surfactants, emulsifiers in food processing, bioimmobilization in nanotechnology, surface application modification and powerful protein purification tags. Many scientists have developed different applications using their self-assembly capabilities. In summary, hydrophobins are vital tools for today's bioengineering and synthetic biology.
BslA, a recently discovered bacterial-origin hydrophobin, has great potential and widely used in synthetic biology. Unlike its fungal-origin cousins, BslA is much easier to produce and has the potential to achieve higher efficiency with low cost.
We constructed fluorescent protein-BsIA fusion genes. EBFP, EGFP, mHoneydew and mOrange were fused to BsIA genes with a GS linker or TEV linker in between, respectively. After induced expression, TEV linker can be cleaved by digestion with TEV enzymes. The reason why we use fluorescent protein as target protein is because they are easy to observe.
We choose pET28a as the expression vector.
We decided to express the fusion protein in E. coli, because E. coli is a mature, low-cost model organism.
We transformed 6 recombinant plasmids (pET28a-EBFP, pET28a-mHoneydew, pET28a-mOrange, pET28a-EBFP-GSlinker-BslA, pET28a-mHoneydew-GSlinker-BslA, pET28a-mOrange-GSlinker-BslA) into BL21 and Rosetta expressing strains.
For 3 recombinant strains (pET28a-EBFP-GSlinker-BslA, pET28a-mHoneydew-GSlinker-BslA, pET28a-mOrange-GSlinker-BslA), TJUSLS_China helped us try three IPTG induction concentrations of 0.1mM, 0.3mM, 0.5mM and two induction temperature of 16°C,37°C, respectively. We found that the induction concentration of 0.5mM IPTG and the induction temperature of 37°C were the best (Figure 2-7a). In addition, the color of the bacterial pellet after centrifugation can also directly prove that the fusion protein of the fluorescent protein has been successfully induced to express (Figure 2-7b).
After the cells of the recombinant strains were induced, centrifuged, and sonicated, the soluble proteins expressed by the strains were all in the supernatant (use 1×PBS as buffer). In order to verify that the fusion protein (EBFP-GSlinker-BslA, mHoneydew-GSlinker-BslA, mOrange-GSlinker-BslA) was successfully fused and expressed compared to the control group (EBFP, mHoneydew, mOrange). We attempted to conduct water contact angle experiments. Due to experimental conditions, we cannot use professional instruments.
We used parafilm as the substrate, which is an extremely hydrophobic interface, and added droplets of the supernatant of the control group and the supernatant of the fusion protein experimental group respectively for observation. We found that the contact angle of the control group was much smaller than that of the experimental group. This means that the supernatant of the control group was hydrophobic as a whole, while the experimental group was hydrophilic. BslA, as a hydrophobin, has the characteristic of reversing surface properties. Through this experiment, we can prove the existence of BslA in the experimental group. (Figure 8)
Then, we used 1×PBS as a blank control, we added isobutanol to the protein supernatant, shaken and let stand for a few minutes until the two phases were clearly separated. We found that the fluorescence color was still in the lower layer (aqueous phase) in both the experimental group and the control group. (Figure 9)
In theory, fluorescence should appear in the upper layer (organic phase) because When a protein fuses with a hydrophobin, the hydrophobin changes the hydrophobicity of the protein, which causes the protein to aggregate into the surfactants.
Our experiments did not get perfect results, we analyzed some possible reasons and tried to continue experiments to explore in the future.
First, the current system is still small. Although we can see the fluorescence color, it is very shallow, and even though there may be some fluorescence in the organic phase, it is not visible to the naked eye due to the small amount. Therefore, we need to expand the system of protein-induced expression in the future.
Second, this may be related to the choice of buffer. We used 1xPBS to dissolve the supernatant obtained after sonication, and it may be possible to change the buffer of other pH to have different results.
Third, it may be related to the hydrophilicity and hydrophobicity of the supernatant. The supernatant contains all proteins expressed by the cells, including the target protein. Through the water contact angle experiment, we can find that the supernatant of the control group is hydrophobic as a whole, which may be caused by the hydrophobicity of some endogenous proteins in cells. Their presence may affect the function of BslA in the ATPS system.
Based on a review published at 2016 (Iqbal,M. et al.), we assumed that other potential rationales that contribute unsuccessful partitioning of protein in ATPS might be unsuitable concentration of salt aqueous solution, unsuitable temperature and incorrect selection of solute in organic phase. Extremely high concentration of salts may alter the hydrophobicity of biomolecules. As a consequence of the hydrophobic ions force the partitioning of counter ions to phase with higher hydrophobicity and vice versa. Thereafter, the addition of salts has critical influence on the partitioning coefficient based on following equation. The temperature can alter the coefficient as well. Moreover, it can generate effect on partitioning through the through viscosity and density.
Conc.AT represents concentration of component A in top phase and Conc.AB represents the concentration of A in the bottom phase at equilibrium.
[1] Hydrophobins: multifunctionalbiosurfactants for interface engineering
https://doi.org/10.1186/s13036-018-0136-1
[2] Continuous Flow Separation of Hydrophobin FusionProteins from Plant Cell Culture Extract
Continuous Flow Separation of Hydrophobin Fusion Proteins from Plant Cell Culture Extract - PubMed (nih.gov)
[3] BslA is a self-assembling bacterial hydrophobin thatcoats the Bacillus subtilis biofilm
www.pnas.org/lookup/suppl/doi:10.1073/pnas.1306390110/-/DCSupplemental.
[4] Aqueous two-phase system (ATPS): an overview and advances in its applicationshttps://doi.org/10.1186/s12575-016-0048-8
