Team:Tianjin/Project
OVERVIEW
In ancient Roman myth, Janus is the god of beginnings and transitions, who is depicted as having two faces, respectively to the future and the past.
Our project is focused on another Janus - hydrophobin the protein, who faces the hydrophilicity and hydrophobicity. Because of this, a sea of new applications are created.
Firstly, we redesign the structures of two classes of hydrophobins, making expression in E.coli possible.
Secondly, we use its double-sticky-tape-like ability to make two applications. We take this advantage to fix antibodies on a high-flux tumor detection chip. Meanwhile, they are used to catch cutinases for plastic degradation. We even make them into a fusion to test if the enhancement could be better.
Thirdly, we use its amphipathicity to achieve protein separation, where they act as a special purification tag, and the system could be as simple as polymer, detergent and water. With help of this, we could even achieve recovery of cutinases.
Feel interested about our story? So let's follow Janus and begin our journey from old to new!
BACKGROUND
As is known to us all, for most proteins, hydrophobic residues are buried in the core of proteins stabilizing the folded conformation of the protein. However, as to our Janus - hydrophobin, one part of the surface is consisted nearly entirely of hydrophobic side chains, forming what was called “the hydrophobic patch” [1]. We could then describe the structure of Janus as a "surfactant" with one hydrophobic and one hydrophilic part.
Janus are proteins that are produced by filamentous fungi, such as Ascomycetes and Basidiomycetes. Many different aspects of fungal development have been attributed to Janus. For example, they are thought to play a role in the formation of aerial hyphae and fruiting bodies. Because Janus could assemble at the medium–air interface and cause the surface tension to be lowered, they could allow hyphae to breach the water–air interface [2].
One of the most important features of Janus is that they are able to assemble spontaneously into amphipathic monolayers at hydrophobic–hydrophilic interfaces [3].What's more, the stability of assembled Janus differs and on the basis of this characteristic two classes of Janus can be distinguished [4]. And the essential differences lie in the occurrence of hydrophilic and hydrophobic amino acid residues i.e. according to their hydropathy plots [1]. Class I Janus generate very insoluble assemblies, which can only be dissolved in strong acids such as trifluoroacetic acid or formic acid, while assemblies of class II Janus can be dissolved in ethanol or sodium dodecyl sulfate or through the application of pressure or lowering of the temperature [5].
References:
[1]M.B. Linder. Hydrophobins: proteins that self assemble at interfaces Curr Opin Colloid Interface Sci, 14 (2009), pp. 356-363
[2]Wo”sten HAB, van Wetter M-A, Lugones LG, van der Mei HC, Busscher HJ, Wessels JGH: How a fungus escapes the water to grow into the air. Curr Biol 1999, 9:85-88.
[3]Wessels, 2000. J.G.H. Wessels. Hydrophobins, unique fungal proteins. Mycologist, 14 (2000), pp. 153–159
[4]Wessels JG: Developmental regulation of fungal cell wall formation. Annu Rev Phytopathol 1994, 32:413-437.
[5]Hydrophobins: Proteins with potential. Hektor H.J., Scholtmeijer K. (2005) Current Opinion in Biotechnology, 16 (4), pp. 434-439.
NEO-PROTEIN DESIGN
Among all kinds of hydrophobins, Class I hydrophobin inJanus (we name it from its insoluble property) from Grifola frondosa and Class II sJanus (we name it from its soluble property) from Trichoderma reesei are models of Class I and Class II respectively. However, it has been reported that bacterial hosts could not be used to produce functional Class I Janus, which causes great obstacles in broader applications of them.
When expressed in E.coli, inJanus usually exist as inclusion body. And there are some possible reasons for this phenomenon: the expression is so fast that protein have no enough time to fold; there are many amino acids with S element, and the environment in E.coli goes against the formation of disulfide bond; lack of necessary enzyme and cofactors [1].
Thus, we made some directed mutations of inJanus, and make its expression in E.coli possible. Meanwhile, we do the same to sJanus. Though it could be expreesed in E.coli (the difference in solubility may lead to it), we would like to research on its difference. Then we made contact angle experiments to test their properties. Ultimately, our project is about four kinds of hydrophobins- inJanus, sJanus and their respective mutants: inJanus-m and sJanus-m.
References:
[1] Zefang Wang. Expression, functional application and self-assembly mechanism of hydrophobin HGF1 (2010).
SUPER PROTEIN CHIP
Background
Protein chip is an emerging technology, which can be used to track the interactions of proteins and to determine their function. Nowadays they are playing a crucial role in various fields around the world, especially in clinical medicine. According to many researches, protein chip has already become a new method of the rapid detection of tumor markers, but it is still a difficult problem to solve the combination between the matrix and the probe, due to the biomolecule inactivation caused by ordinary substrate material.
For instance, polystyrene, which has hydrophobic surface, is a commonly used substrate. When antibodies are absorbed onto it, the strong hydrophobic force between them lead to the formation of multilayer film. Among all the antibodies that are absorbed, only the top of them can maintain their activities, while the rest would go through conformational changes and become inactivated. This would result in low sensitivity and a huge waste of reagents.
Scientists have always tried to find new method of surface modification to improve substrate properties, such as three-dimensional surface modification of glass or use gold membrane to replace the ordinary substrate. However, these methods show either complex operation or high cost[1].
A very interesting characteristic of assembled Janus is the amphipathic nature of the coating. By changing the hydrophobicity of a surface the binding of various molecules and cells can be manipulated [2]. In view of this, Janus could find use in substrate modification applications.
Our Goal
Based on previous experience, we use Janus to modify protein chip substrate so as to optimize it. In the process of our experiment, Janus acts as a medium in antibody-fixing by electrostatic force [3]. Compared to the traditional methods, this fixing method not only ensures the activity of the antibody, but also improves the detection’s accuracy rate. Additionally, it can greatly reduce the cost of chip’s making [4]. The most important thing is that this method enables us to use antigens in very light concentration, which can save a lot of samples.
Our Design
In our experiment, we used two kinds of Janus-inJanus and sJanus. inJanus generate more stable assemblies compared to sJanus. We use hydrophobins from two categories on purpose of studying the differences between them in aspect of surface modification.The serum levels of tumor markers are directly related to the state of cancer. The quantitative detection of them in serum will be valuable for clinical research and early diagnosis [5]. In our experiment, we chose CEA, AFP and CA15-3 and their respectively antibodies as samples. Levels of CEA can be associated with lung cancer, and AFP - liver cancer, CA15-3 - breast cancer. In fact, there are a large number of tumor markers that associated with cancer, but the markers we chose are more representative and easier to purchase.
Based on Double Antibody Sandwich Method, we built a biosensor system which is suitable for the measurement of a wide range of biomarkers. Though the specific binding of antigen (tumor marker) and their polyclonal antibody, we can use the fluorescence microscope to detect the signal and analyze so as to get the test results, then, we can judge the type of tumor.
Our project fully takes advantage of the high-throughput of the protein microarray, the high-specificity of the Double Antibody Sandwich Method [6], the hypersensitivity of the fluorescence labeling, in order to detect tumor markers rapidly and precisely, and discover patients’ condition in time, having quite vast potential for future medicinal development.
References:
[1] Knoll W,Liley M,Piscevic D,et a1.Supramolecular architectures for the functionalization of solid SUrfaces[J].Adv Biophys,1997,34
[2] Wessels JG: Hydrophobins: proteins that change the nature of the fungal surface. Adv Microb Physiol 1997, 38:1-45.
[3] Chunwang Peng, Jie Liu, Daohui Zhao, and Jian Zhou : Adsorption of Hydrophobin on Different Self-Assembled Monolayers: The Role of the Hydrophobic Dipole and the Electric Dipole. School of Chemistry and Chemical Engineering, Guangdong Provincial Key Lab for Green Chemical Product Technology, South China University of Technology, Guangzhou, Guangdong 510640, P. R. China.
[4] Bayry J, Aimanianda V, Guijarro JI, Sunde M, Latge ´ JP (2012) Hydrophobins—Unique Fungal Proteins. PLoS Pathog 8(5): e1002700. doi:10.1371/journal.ppat.1002700
[5]. Miao, X., Zou, S., Zhang, H., Ling, L., 2014. Sens. Actuators B 191, 396–400.
[6] Markus F. Templin, Dieter Stoll, Monika Schrenk, Petra C. Traub, Christian F. Vöhringer and Thomas O. Joos: Protein microarray technology.TRENDS in Biotechnology Vol.20 No.4 April 2002
STIMULATED PLASTIC ENZYMOLYSIS
Background
Since completely synthetic plastic materials came out in 1900s, it was used more and more extensively due to its cheapness and durability. Nowadays, plastics has covered our lives in many areas. However, it is slow to degrade, which has led to serious plastic pollution.
Plastic reduction efforts have occurred in some areas in attempts to reduce plastic consumption and pollution and promote plastic recycling. Scientists have come up with many methods in order to solve white pollution, such as landfill, incineration and chemical decomposition. It is a good idea to degrade plastics by enzymes. Compared with the traditional physical and chemical methods, it costs lower power and is more environmental. However, biological method has much lower degradation efficiency. Therefore, improving the efficiency of biological plastic degradation has no time to delay.
Plastic degradation is not only of vital importance for solving environmental pollution. For example, enzymatic recycling of PET basically would break down the polymer into its building blocks ethylene glycol (EG) and terephthalic acid (TA), which have a high value and can be reused in chemical synthesis, including the production of PET. This would avoid current limitations in plastic recycling, which requires pure plastic fractions or has to fight with enrichment of contaminants [1]. At the same time, it provides a new method for plastic surface modification, making it possible for applying finishing compounds and coloring agents.
Among all kinds of plastics, PET (polyethylene terephthalate) plays a major role. It is the most commonly used synthetic fiber (25 million tonnes produced annually worldwide) and is forecasted to account for almost 50% of all fiber materials in 2008[2]. PET hydrolysis can be achieved by enzymes from distinct classes, such as esterases, lipases, and cutinases, the latter thereby yielding the most promising results [3]. Hence, we choose three kinds of cutinases for our project – FsC (Fusarium solani cutinase), LC (leaf-branch compost cutinase) and Thc_Cut1 (Thermobifida cellulosilytica cutinase). Especially, FsC was used and by TU_Darmstadt in 2012 iGEM, and LC by UC_Davis in 2012 iGEM.
Our Goal
1. Stimulate PET hydrolysis by FsC, LC and Thc_Cut1 with addition of four kinds of Janus.
2. Enhance enzyme activity by making fusion proteins with Janus.
3. Compare the differences between four kinds of Janus for stimulating cutinase activity.
4. Study the mechanism behind Janus's acceleration.
Our Design
In 2005, Toru Takahashi etc. found that growth of the mold fungus Aspergillus oryzae on the biodegradable polyester polybutylene succinate-coadipate (PBSA) induced not only the cutinase (CutL1) but also a hydrophobin (RolA), which adsorbed to the hydrophobic surface of PBSA, recruited CutL1, and stimulated its hydrolysis of PBSA [4]. This gives us the inspiration that Janus may stimulate enzymatic plastic degradation. As well, in 2013, Liliana Espino-Rammer etc. added two kinds of Class II hydrophobins HFB4 and HFB7 of Trichoderma into the process of PET hydrolysis by Humicola insolens cutinase, and they turned out to stimulate the enzyme activity [4].
In our project, we would try two new hydrophobins, inJanus and sJanus, and their respective mutants, to test if they have the ability to increase the activity of above-mentioned three cutinases- FsC, LC and Thc_Cut1. It is the first time to study two classes of Janus in the process of plastic degradation, and it could reveal the differences between them and provide a better hydrophobin partner. Meanwhile, will our brand new inJanus-m and sJanus-m behave well in the process? We would like to explore.
Because the mechanism behind Janus' acceleration is still unknown, we would try different ways to add Janus into plastics, and various ways could lead to huge difference of Janus's effect. The first way is simple mixing, which means mixing cutinase, Janus and PET at the same time. The second way is PET pre-incubation with Janus, which means adding Janus into plastics primarily, and then putting cutinase in the container after 24 hours. The third way is making Janus and cutinase into fusion protein, which is a novel work that could increase cutinases' activity radically.
Furthermore, we develop comprehensive and innovative models on Janus-enhanced plastic enzymolysis. We make a good combinaton of thermodynamics and dynamics, and a novel kinetic model is built to better describe the enzyme catalyzed hydrolysis of PET by combing the Michaelis-Menten equation with the Langmuir equation. Meanwhile, we propose models to describe the effect of Janus into the hydrolysis system based on the hyphothetical elementary reactions using the result of self-asembling model and the novel kinetic model in the first part. Thirdly, to visualize the result of our modelling, we write a program using MATLAB which simulates PET degradation process. (Click here to see more about our models!)
References:
[1] Espino-Rammer L, Ribitsch D, Przylucka A, Marold A, Greimel KJ, Herrero Acero E, Guebitz GM, Kubicek CP, Druzhinina IS. 2013. Two novel class II hydrophobins from Trichoderma spp. stimulate enzymatic hydrolysis of poly(ethylene terephthalate) when expressed as fusion proteins. Appl. Environ. Microbiol. 79:4230-4238.
[2] Guebitz, G.; Cavaco-Paulo, A. Enzyme go big: surface hydrolysis and functionalisation of synthetic polymers. Trends Biotechnol. 2007, 26 (1), 32–38.
[3] Eberl A, Heumann S, Kotek R, Kaufmann F, Mitsche S, Cavaco-Paulo
A, Gübitz GM. 2008. Enzymatic hydrolysis of PTT polymers and oligomers. J. Biotechnol. 135:45–51.
[4] Takahashi T, Maeda H, Yoneda S, Ohtaki S, Yamagata Y, Hasegawa F,
Gomi K, Nakajima T, Abe K. 2005. The fungal hydrophobin RolA recruits polyesterase and laterally moves on hydrophobic surfaces. Mol. Microbiol. 57:1780–1796.
PROTEIN EXTRACTION KIT
Background:
For selective purification of proteins, affinity chromatography is one of the most efficient methods available. Often they are only suited for analytical purposes and purification of high-value products and are too expensive for large-scale products such as industrial enzymes. Furthermore, the methods are difficult to scale up [1].
Liquid-liquid extraction in an aqueous two-phase system has been applied for primary recovery of industrial bulk proteins. They are formed in mixtures between two incompatible components, e.g. PEG/dextran, polymer/salt or detergent/polymer.
Usually, aqueous two-phase systems' formation could be induced by a shift in temperature above a critical temperature (cloud point) in a detergent/water system. Non-ionic detergents, such as Triton X-114, display such temperature-sensitive phase separation, and can form cloud point extraction (CPE) systems [2].
The partitioning of a protein to one of the phases may depend on its surface charge or hydrophobicity, but the driving forces are not well understood. Obviously, the selectivity and overall efficiency of purification depend on how much the target protein differs in relevant properties as compared to the rest of proteins in the mixture. To achieve selective protein purification in aqueous two-phase system, small hydrophobic tags containing tryptophans have been fused to the target protein. However, the production of these fusion molecules is not efficient, possible reasons being problems in secretion of the tagged proteins or proteolytic degradation of the tag [1].
Our Goal
1. Achieve efficient protein extraction using Janus tag.
2. Find the best Janus tag.
3. Testify this system applicative to all kinds of proteins.
4. Develop universal BioBrick Janus tag for all users.
5. Explore the universally good conditions to all kinds of proteins.
6. Propose standard protocols for Janus-based protein extraction.
Our Design:
Here comes the Janus, which could perform as a perfect protein extraction tag. The size of inJanus is about 8 kDa, and sJanus is about 7.5 kDa; both of them are not very large to affect the target protein. At the same time, Janus could promote the production of some proteins in various cells, like E4GIcore-sJanus in T. reesei [2] and GFP-sJanus in Nicotiana benthamiana plants [3].
To purify the target protein, we would make the target protein and Janus into a fusion protein, which is connected with a linker. Then, we could use aqueous two-phase system to make them separated from the bulk protein, according to the property that Janus will direct to the phase of detergent in the system of detergent/water.
In our project, we would use four kinds of Janus to find the best one. Meanwhile, we aim to design a standard protocol to make this system applicative to almost every kind of protein, and we call it Protein Extraction Kit.
Firstly, we use the principle of RFC 23 to design a universal Janus tag, which includes a linker able to be cut by TEV protease and a sJanus gene. Thus, if other teams want to use this system to express and purify their proteins, they could use the standard assembly to make a fusion protein. Secondly, we explore for the best detergent, the best concentration of detergent and the best buffer solution. Thirdly, we construct many fusion proteins, including three kinds of fluorescent proteins (GFP, BFP and RFP) with four kinds of Janus, which are inJanus, sJanus and their respective mutants. We also construct experiments about fusion proteins with cutinases Thc_Cut1, FsC and LC to test if this system could be used on the putification of enzymes. Furthermore, we build complete models to testify that this system is applictive to all kinds of protein and search for the universally good conditions for almost every protein.
Of course, we also conduct experiments to detect the efficiency of this method and make sure that the target proteins have not lost their activity.
References:
[1] Linder MB, Qiao M, Laumen F, Selber K, Hyytiä T, Nakari-Setälä T, Penttilä ME (2004) Efficient purification of recombinant proteins using hydrophobins as tags in surfactant-based two-phase systems. Biochemistry 43:11873–11882
[2] Collen, A., Persson, J., Linder, M., Nakari-Setälä, T., Penttilä, M., Tjerneld, F., Sivars, U., 2002. A novel two-step extraction method with detergent/polymer systems for primary recovery of the fusion protein endoglucanase I-hydrophobin I. Biochimica et Biophysica Acta (BBA) 1569, 139–150.
[3] Joensuu JJ, Conley AJ, Lienemann M, Brandle JE, Linder MB, Menassa R. Hydrophobin fusions for high-level transient protein expression and purification in Nicotiana benthamiana. Plant Physiol 2010;152:622-33.
Protein Expression
The first step of our experiments is to get the target proteins. And we have expressed what we need successfully.
Neo-Protein Design
Pre-expression:
The bacteria were cultured in 5mL LB liquid medium with kanamycin in 37℃ overnight. After taking samples, we transfer them into 1L LB medium with kanamycin.
Cultured in bottles:
After 4 hours culturing in 37℃ in bottles, we used 500μM IPTG induced in 16℃ for 8-12h. We used McAc 0 and 20 to wash off the bulk proteins, and used McAc 200 to wash off aimed proteins.
Figure 1. The expression of inJanus-m
We finally can use McAc 0 and 20 to wash off the bulk proteins and use McAc 200 to wash off aimed proteins.
Figure 2. The expression of sJanus-m
The concentration of IPTG, temperature and time we used in sJanus-m’s expression are same to those of inJanus-m, except that we used TB liquid culture medium rather than LB to raise bacteria to get more proteins.
We also expressed sJanus successfully in E.coli (data not provided). And Nankai helped us to express inJanus in pichia pastoris, which you could see in detail in (COLLABORATIONS).
Stimulated Plastic Enzymolysis
1. About FsC
Inoculate of 1L liquid LB-media with C+_FsC-sJanus_pET-28a, C+_FsC-sJanus-m_pET-28a for 4h. Add 1M IPTG 500μL and induce at 16°C for 15h. Centrifuge of 1L LB medium at 4000rpm for 20min and discard supernatant. Crush bacteria with High Pressure Homogenizer and centrifuge at 18000rpm for 30min. Then purify protein with nickel column and we get the FsC-sJanus and FsC-sJanus-m.
Figure 3. The result of protein C+_FsC-sJanus expression. M is Protein marker. a is sample of FsC-sJanus in C+ which is non-induced. b is sample of FsC-sJanus in C+ which is induced. c is sample of sediment after breaking bacteria and centrifugation. d if sample of liquid after filtration by Ni column. e is sample of media after filtration by Ni column. f is sample of liquid after removing impurity with 50mM MCAC. g is sample of media after removing impurity with 50mM MCAC. h is sample of liquid after washing with 200mM MCAC. i is sample of media after washing with 200mM MCAC. j is sample of target protein after washing with 200mM MCAC.
Figure 4. The result of protein C+_FsC-sJanus-m expression. M is Protein marker. a is sample of FsC-sJanus-m in C+ which is non-induced. b is sample of FsC-sJanus-m in C+ which is induced. c is sample of sediment after breaking bacteria and centrifugation. d if sample of liquid after filtration by Ni column. e is sample of media after filtration by Ni column. f is sample of liquid after removing impurity with 50mM MCAC. g is sample of media after removing impurity with 50mM MCAC. h is sample of liquid after washing with 200mM MCAC. i is sample of media after washing with 200mM MCAC. j is sample of target protein after washing with 200mM MCAC.
2. About Thc_Cut1
Protein pre-expression experimental conditions:
1. Transferred our correct plasmid into escherichia coli BL21 (DE3).
2. Select bacterial colony and add into LB, incubate at 37 centigrade for 7h. Add 4μl of IPTG to induce the expression of protein.
3. Incubate at 37 centigrade for 4h. The bacterial solution’s OD is 0.6-0.8.
Figure 5. The result of protein Thc_Cut1 pre-expression. 1: bacterial No.1 non-incubated; 1’: bacterial No.1 incubated; 2: bacterial No.2 non-incubated; 2’: bacterial No.2 incubated; 3: bacterial No.3 non-incubated; 3’: bacterial No.3 incubated
Figure 6. The result of protein Thc_Cut1-sJanus-m pre-expression. Labels are the same as before.
Figure 7. The result of protein Thc_Cut1-sJanus pre-expression. Labels are the same as before.
Finally, the protein was expressed successfully.
We added 5μl of the bacterial restored into the LB containing 5μl of ampicillin to the final concentration of 1mM. And then, we incubated them in the shaker at the temperature of 37 centigrade, working for 14-16 hours.
Experimental conditions as follow：
1. Incubate at 37 centigrade, until OD ranges from 0.6-0.8(4-5h).
2. Incubate at 4 centigrade, 220rpm, for 30mins.
3. Add 1mL IPTG to the final concentration of 1mM.
4. Incubate at 16 centigrade for 12-16h.
The purification of protein:
We used eppendorf to make bacterial deposited, working at 4000rpm for 20min. Use 15mL MCAC0 to suspend bacterial.
After resuspending, we used high pressure to break the cells.
In order to get the recombinant protein with the higher purity, the recombinant protein was purified through Ni-chelating affinity chromatography.
The results are as follow:
Figure 8. The result of protein Thc_Cut1 expression. 1: Sample non-induced; 2: Sample induced; 3: Sample of cytoplasm deposited; 4: Sample which outflowed from Ni column combined with supernatant; 5:Sample of NIi medium combined with supernatant; 6: Sample washed by MCAC20; 7: Sample resuspended by MCAC30; 8: Sample washed by MCAC30; 9: Sample resuspended by MCAC50; 10: Sample washed by MCAC50; 11: Sample resuspended by MCAC100; 12: Sample washed by MCAC100; 13: Sample resuspended by MCAC200; 14: Sample washed by MCAC200; 15: Sample resuspended by MCAC500; 16: Sample washed by MCAC500; 17: Sample resuspended by MCAC1000.
Figure 9. The result of protein Thc_Cut1-sJanus expression. 1: Sample non-induced; 2: Sample induced; 3: Sample of cytoplasm deposited; 4: Sample of supernatant; 5: Sample of NIi medium combined with supernatant; 6: Sample which outflowed from Ni column combined with supernatant; 7: Sample washed by MCAC20; 8: Sample resuspended by MCAC30; 9: Sample washed by MCAC30; 10: Sample resuspended by MCAC50; 11: Sample washed by MCAC50; 12: Sample resuspended by MCAC100
Figure 10. The result of protein Thc_Cut1-sJanus expression.13: Sample washed by MCAC100; 14: Sample resuspended by MCAC200; 15: Sample washed by MCAC200; 16: Sample resuspended by MCAC500; 17: Sample washed by MCAC500; 18: Sample resuspended by MCAC1000.
Figure 11. The result of protein Thc_Cut1-sJanus-m expression. 1: Sample non-induced; 2: Sample induced; 3: Sample of cytoplasm deposited; 4: Sample which outflowed from Ni column combined with supernatant; 5: Sample of NIi medium combined with supernatant; 6: Sample washed by MCAC20; 7: Sample resuspended by MCAC30; 8: Sample washed by MCAC30; 9: Sample resuspended by MCAC50; 10: Sample washed by MCAC50; 11: Sample resuspended by MCAC100; 12: Sample washed by MCAC100; 13: Sample resuspended by MCAC200; 14: Sample washed by MCAC200; 15: Sample resuspended by MCAC500; 16: Sample washed by MCAC500; 17: Sample resuspended by MCAC1000
Protein Extraction Kit
Besides cutinase-Janus fusion proteins, we also expressed fluorescent protein- Janus fusion for this project.
1. About GFP
GFP-sJanus:
Figure 12. The result of protein GFP-sJanus expression. In this picture of gel, we can see that when induced by IPTG, the gene of GFP-sJanus expressed. The correspond place has been underlined by red line.
Figure 13. The result of protein BL21_GFP-sJanus expression. H is Protein marker. G is sample of liquid after filtration by Ni column. F is sample of media after removing impurity with 20mM MCAC.E is sample of liquid after removing impurity with 20mM MCAC. D is sample of media after removing impurity with 50mM MCAC. C is sample of liquid after removing impurity with 50mM MCAC. B is sample of media after removing impurity with 100mM MCAC.A is sample of liquid after removing impurity with 100mM MCAC.
Figure 14. The result of protein BL21_GFP-sJanus expression. A is Protein marker. B is sample of media after washing with 100mM MCAC. C is sample of liquid after washing with 100mM MCAC. D is sample of media after washing with 200mM MCAC. E is sample of target protein after washing with 200mM MCAC. F is sample of media after washing with 500mM MCAC. G is sample of target protein after washing with 500mM MCAC. H is sample of target protein after washing with 1000mM MCAC.
After that we use the target protein concentrating, we got our target protein GFP-sJanus, whose concentration is 50.1mg/ml.
GFP-sJanus-m:
Figure 15. The result of protein GFP-sJanus-m expression. In this picture of gel, we can see that when induced by IPTG, the gene of GEP-sJanus-m expressed. The correspond place has been underlined by red line.
Figure 16. The result of protein BL21_GFP-sJanus-m expression. G is sample of liquid after filtration by Ni column. F is sample of media after removing impurity with 20mM MCAC.E is sample of liquid after removing impurity with 20mM MCAC. D is sample of media after removing impurity with 50mM MCAC. C is sample of liquid after removing impurity with 50mM MCAC. B is sample of media after removing impurity with 100mM MCAC.A is sample of liquid after removing impurity with 100mM MCAC.
Figure 17.The result of protein BL21_GFP-sJanus-m expression. A is Protein marker. B is sample of media after washing with 100mM MCAC. C is sample of liquid after washing with 100mM MCAC. D is sample of media after washing with 200mM MCAC. E is sample of target protein after washing with 200mM MCAC. F is sample of media after washing with 500mM MCAC. G is sample of target protein after washing with 500mM MCAC. H is sample of target protein after washing with 1000mM MCAC.
After that we use the target protein concentrating, we got our target protein GFP-sJanus-m, whose concentration is 26.7mg/ml.
We also expressed GFP-inJanus and GFP-inJanus-m fusion protein (data not provided).
2. About BFP
We insert the plasmid into competent C+ to make the fusion protein secreted.
Express the fusion protein:
|
5ml tube with C+ |
37℃ 15h |
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1L culture bottle |
37℃ 4h |
|
induced by IPTG |
500μl 16℃ 12-16h |
|
Collect the bacterial |
|
|
Break the bacterial |
Purify the fusion protein:
|
0mM McAc |
Resuspension |
|
10mM McAc |
Wash the impurity away |
|
20mM McAc |
Wash the impurity away |
|
30mM McAc |
Wash the impurity away |
|
50mM McAc |
Wash the impurity away |
|
200mM McAc |
Elute |
Figure 18. BFP- sJanus: The target protein is about 40kDa. As is shown in the picture, the protein is purified through Ni-chelating affinity chromatography.
Figure 19. BFP- sJanus-m: The target protein is about 40kDa. As is shown in the picture, the protein is purified through Ni-chelating affinity chromatography.
1. About RFP
Transforming:
Transformed the expression vectors(concentration:121.7ng/mL) we had already built before (pET-28a) into the Escherichia coli BL21, then incubated the bacteria for 12h (37℃).
Pre-expression:
Picked up the single bacteria colony on the culture dish by a tip and put it into a 5mL LB liquid medium, incubated in the shaking table for 6h (37℃), took 400μL from it for Glycerol Stocking (-80℃) and took 200μL centrifuge and the supernatant was discarded, 20μL ddH20 re-suspended and then made sample for glue leaking, added 5μL IPTG into the rest and incubated for 4h, took 200μL for centrifuging and the supernatant was discarded, 20μL ddH20 re-suspended and then made sample for glue leaking. The leaking result is as followed.
Figure 20. The result shows that after induction the bacteria expressed obviously large amount of the target protein (length:30~40)
Expression
Added 5μL Glycerin bacteria into a 5mL LB liquid medium and incubated about 15h, then added them into the 1L LB liquid medium, incubated under 37℃ until the OD was 0.6~0.8(about 4h), then cooling the atmosphere to 4℃ and incubated 0.5h, added 1mL IPTG into the flask and incubated for 16h under 16℃.
Figure 21. The expression of RFP fusion protein. The color is very obvious
Purification:
Centrifuged the mixture 4000rpm for 30min, then re-suspended the sediment by 15mL McAc. Used the ultrasonication to destroy the bacteria, then centrifuged 18000rpm for 30min and purred the supernatant by Ni-NTA, took the sample of the sediment; mixture, McAc 20, 30, 50, 100, 200, 500 which was combined with medium; misture, McAc 20, 30, 50, 100, 200 which leaked out of the Ni-NTA. Then made sample for glue leaking. The leaking result is as follow.
Figure 22.According to the result, the best removing impure concentration is 30, the best elution concentration is 200, and we got the pure protein after McAc 200 elution.
STIMULATED PLASTIC ENZYMOLYSIS
Pre-experiment
overview
We have found out appropriate concentration of the cutinase we made and suitable pH for our enzyme Thc-Cut1, the right time to detect the product of hydrolysis of PET.
Result
We use absorbance in 600 nm to measure the turbidity of the liquid in tube. We tried different concentration of enzyme with one piece of plastic, decided to use 2mg/ml to conduct our experiment afterwards.
Figure 1. This curve describe the OD600nm after 3h’s reaction changes through the concentration of Thc_Cut1. We can see clearly at 2mg/ml ,the curve reaches a peak, at which concentration we will compare the hydrolysis effect.
We also conducted our system in various pH, found that the activity of Thc_Cut1 doesn’t change much in different pH, so we chose pH 7.0 as one of our experimental condition.
Figure 2. The curve shows the absorbance at 600nm changes along with pH. The activity of enzyme doesn’t change much in various pH.
In order to find right time to detect, we detect OD600nm in several time, here’s the time line.
Figure 3. The timeline clearly indicates the hydrolysis rate rises along with time.
Considering we need an appropriate detect time to measure the hydrolysis effect, we set our detect time at 3h.
Main experiment
Overview
We discovered the right temperature to conduct various way to mix up our sJanus (or inJanus) and Thc_Cut1. And we have found out the best way to increase the hydrolysis rate.
Result
We conduct our trail in different temperature, realizing 50℃ is the proper temp for the enzyme and our Janus to combine.
Figure 4. Different color represent different ways of mix. Thc_Cut1+Janus means enzyme had incubated with Janus for 16h before the plastic was added into the system. And Plastic+Janus means plastic had incubated with Janus before adding enzyme. And we also conduct these incubation separately in 4℃,37℃.50℃, values at 50 degree are higher than that under the other temperature.
Based on the experiment above, we compared these two different ways with simple mix, here’s the result.
Figure 5. The tag with “*” means simple mixture.
We can conclude that pre-incubation does increase the hydrolysis rate, some ways(Plastic+sJanus) even boost 200%+ react rate comparing with Thc_cutL1* in our conditions!! Another interesting phenomenon is sJanus usually works better than inJanus, and the reason behind it need our further exploration. On the whole, Our Janus works a lot!
Further exploration
Overview
We successfully build a fusion protein which attach cutinase Thc_Cut1 to our Janus (sJanus-m).
The fusion protein has much more better performance than the cutinase before.
Result
The procedure in this part is likely to the pre-experiment, we use different concentration of fusion protein. And we compare the data to the value we conduct with Thc_Cut1 before, found that fusion protein hydrolysis more, especially in high concentration.
Figure 6. These curves compare fusion protein’s performance with normal cutinase in different concentration. We can see that fusion protein perform much better when concentration are over 1mg/ml.
We also conduct our experiments in different pH by using different Tris-HCl. Just as picture shows, the activity of our fusion protein doesn’t change a lot in different pH which is similar with Thc_Cut1.
Figure 7. These two proteins are stable when pH changes, especially when pH is between 6.0 and 9.0. And the concentration of Thc_Cut1 and Thc_Cut1-sJanus-m are 0.2mg/ml.
We drew a curve about detect time and compared it with Thc_Cut1, we can see the hydrolysis rate rose sharply after 5h,while Thc_Cut1 still in the low level. Which represent our Janus works well!
And our fusion protein does improve a lot!
Figure 8. We detect absorbance of liquid every 3h, red line refers to our fusion protein, and the blue line is former enzyme. The performance of cutinase are highly improved when fuse to the sJanus-m.
Figure 9 &10. The picture shows 5mg/ml enzyme have react with one piece of plastic（3*3*0.25mm）for 3h, before and after centrifugation. We can see clearly in the pic. After 3hs the tube of Thc_Cut1-sJanus-m had much more sediment than Thc_Cut1.
PROTEIN EXTRACTION KIT
Background
ATPS (aqueous two-phase systems) is a novel technology to purify proteins. The mechanism of this system lies in the partition between two different phases.
Aims
Construct a brand new and standard way to purify proteins based on aqueous two-phase system.
Results
1. Confirm the strengths using aqueous two-phase systems
2. Successfully separate the target proteins from bulk protein phase based on aqueous two-phase systems at a high partition rate.
3. Construct a standard protocol to separate different kinds of proteins.
The experiment of ATPS
Pre-experiment
Process of this experiment
In the pre-experiment, the original concentration of our protein is about 50ug/ml, which volume is 200uL. We designed the pre-experiment just in order to make preparations for our next experiment. We added 5% (v/v) Berol 532 to protein solution. And then, we used shaker to make them mixed at the speed of 250r/min and 20 centigrade working about one hour. Centrifuge was used to make them separated and came into being two phases at the speed of 8000g for about 25min. In these two phases, the upper phase is rich-detergent and the lower phase is depleted-detergent phase. Because of the property of hydrophobin, fusion protein will stay in the detergent phase, and bulk protein stay in the water phase. We put the rich-detergent phase in another centrifuge cubes and added butanol which is 5 times volume of detergent. Centrifuge was used to make them separated and finally in the lower phase (water phase), we got pure target protein. The upper phase contains detergent (Berol 532), which can be recycled.
Reagents used in this experiment
|
Concentration(final) |
Volume |
|
|
Berol 532 |
Purity 96% |
10uL (5%) |
|
Protein solution |
50ng/mL 100ng/mL 3mg/mL |
200uL |
Results of this experiment
Figure 1. In this picture of gel, the third lane is the original protein solution and the forth is the protein solution which used ATPS to purify. We can see that some bulk proteins have been removed. This fusion protein is GFP-inJanus. The first and second lane are GFP-inJanus, because we didn’t dilute the protein’s solution, the concentration of protein is so high (about 3mg/mL) that they overflowed. The next is 100ug/mL and 50ug/mL.
Figure 2. We used Bandscan5.0 to analysis the gel. We can see that other proteins have been removed all in the third and fourth lane. Meanwhile, we can see that other proteins have been removed to some degree in the fifth and sixth lane.
We calculated the efficiency of this experiment. It is 18.93% in the third and fourth lane and 46.17% in the fifth and sixth lane. The efficiency is a bit low and we analyzed the reasons. We added 10uL of Berol 532 in this system to extract the target protein. However, the total amount of protein is so large that our detergent can’t extract them all.
Formal experiment using Berol 532 as detergent and MCAC0 as buffer
Process of this experiment
In our first formal experiment, we changed some conditions. We increased the speed of shaker to 300r/min in order to make them mixed totally. Meanwhile, in the pre-experiment, we found that we didn’t need to use centrifuge to make them separated because they can divided into two phases automatically in few seconds at the room temperature(Berol 532, article says that its low solubility does not allow cloud point measurement). We used 20% (v/v) Berol to extract our fusion proteins. If less detergent used in this system, it will increase the final concentration of proteins, but it will decrease the efficiency of extraction because detergent is lacking in combing the fusion protein.
The volume of our system is about 5mL (We used original solution which got by centrifuge and high pressure after suspending by MCAC0). We added 1mL Berol 532(20% w/w) in our original solution. And then, we used shaker to make them mixed at the speed of 300r/min and 20 centigrade working about one hour. We put the tube at the room temperature and the mixture was separated into two phases in few seconds. We got the upper phase (2mL) and added water saturated butanol (5mL). Then we used centrifuge to make them separated at 4 centigrade (In this temperature, we can prevent protein from denaturation for a little longer time) at the speed of 3500rpm (8000g and 3500rpm both worked) working for 10 minutes. We got the lower phase, and it contains our target protein. We did this twice in order to increase the purity of our target proteins. We used technology of SDS-PAGE to test if this system works.
Reagents used in this experiment
|
Concentration(final) |
Volume |
|
|
Berol 532 |
Purity 96% |
1mL (20%) |
|
Protein solution |
Not detect by machine, but we can analyze by gel. |
5mL |
Results of this experiment
Figure 3. In this picture of gel, the first lane is the original solution of protein. We can see that it contains many other proteins. The second lane is the lower phase (water phase), we can see that the target protein have been removed and some bulk proteins stay in the water phase. The third and fourth lane are the water phase got in the reverse extraction. We can see that the target proteins have been concentrated and some other proteins have been removed.
Figure 4. This picture was shot in ultraviolet, we can see clearly that our target protein stay in the detergent phase because of the blue fluorescence.
Figure 5. We used Bandscan5.0 to analysis the gel. We can see that target proteins have been purified by ATPS. In this process, other proteins stay in the water phase and our target protein stay in the detergent phase. When extracted by saturated butanol, target protein was concentrated in the water phase. The picture of gel shows that when used it twice, the purity will increase again
We calculated the efficiency of this experiment. It is 67.54% in the twice and fourth lane and 75.06% in the fourth and fifth lane. The efficiency of it is high to some degree, but we want get a higher separation rate and increase the purity of our target protein. In our next experiment, we used many buffer and different detergent to achieve it.
Formal experiment using Berol 532 as detergent and HAc/NaAc as buffer
The process of this experiment
In this experiment, the fusion proteins we used are GFP-inJanus and BFP-inJanus. We added 2%, 5% of Berol 532 in GFP-inJanus and 2%, 5% (v/v) of Berol 532 in BFP-inJanus. We added 200uL HAc/NaAc(pH=7.5) to 4800uL protein solution. And then, we used shaker to make them mixed at the speed of 300r/min and 20 centigrade working about one hour. We added butanol which is 5 times volume of detergent to do reverse extraction. Centrifuge was used to make them separated and finally in the lower phase (water phase), we got pure target protein.
Reagents used in this experiment
|
Concentration(final) |
Volume |
|
|
Berol 532 |
Purity 96% |
100uL(2%) 250uL(5%) |
|
Protein solution (GFP-inJanus BFP-inJanus) |
Not detect by machine, but we can analyze by gel. |
200uL protein solution+4800uL buffer |
Results of this experiment
Figure 6. In this picture of gel, the first lane is marker. The second lane is the original solution of GFP-inJanus. The third lane is the system adding 2% and the fourth is that adding 5%. We can see clearly that other proteins have been removed. Unfortunately, because of some unknown reasons, there isn’t any line about BFP-inJanus in this gel.
Figure 7. We used Bandscan5.0 to analysis the gel. The percentage of our target protein in total proteins is 11.5% before separating. It increased to 59.1% in the third lane(2% Berol 532) and 88.7% in the fourth lane(5% Berol 532). Many other proteins have been removed.
Figure 8. We can see the green florescence in the upper phase. This picture was shot in ultraviolet.
We did the second group of experiment. In this experiment, the concentration of protein solution and volume of Berol 532 changed. The total volume of protein solution is 6mL. The conditions doing this experiment is same as above.
Reagents used in this experiment
|
Concentration(final) |
Volume |
|
|
Berol 532 |
Purity 96% |
120uL (2%) 300uL (5%) |
|
Protein solution (GFP-inJanus) |
GFP-inJanus (0.794mg/mL) |
100uL protein solution+5900uL buffer |
Results of this experiment
Figure 9. In this picture of gel, the first lane is original protein solution. The second and third lane are proteins separated by ATPS. The former is 2% of Berol 532; the latter is 5% of Berol 532. We can see that the system added 5% of Berol 532 get the best result.
Figure 10. We used Bandscan5.0 to analysis the gel. The percentage of our target protein in total proteins is 26.4% before separating. It increased to 74.6% in the third lane(5% Berol 532). Many other proteins have been removed. However, there isn’t any line detected in the second one.
We did the third group of experiment. In this experiment, the protein we used is RFP-inJanusm. The conditions doing this experiment is same as the second group.
Reagents used in this experiment
|
Concentration(final) |
Volume |
|
|
Berol 532 |
Purity 96% |
120uL(2%) 300uL(5%) |
|
Protein solution (RFP-inJanusm) |
RFP-inJanusm (0.702mg/mL) |
100uL protein solution+5900uL buffer |
Results of this experiment
Figure 11. In this picture of gel, the first lane is original protein solution of RFP-inJanusm. The second and third lane are proteins separated by ATPS. The former is 2% of Berol 532; the latter is 5% of Berol 532. We can see that the system added 5% of Berol 532 get the best result.
Figure 12. We used Bandscan5.0 to analysis the gel. The percentage of our target protein in total proteins is 19.5% before separating. It increased to 78.6% in the second lane(2% Berol 532) and 60.9% in the fourth lane(5% Berol 532). Many other proteins have been removed. This experiment confirmed that RFP-inJanusm does work.
We did the fourth group of experiment. In this experiment, we used GFP-inJanus, BFP-inJanus, RFP-inJanus and RFP-inJanusm to test this system. The concentration of original protein solution was detected by BCA measurement. The conditions doing this experiment is same as the second group.
Figure 13. We did many groups of experiment.
Reagents used in this experiment
|
Concentration(final) |
Volume |
|
|
Berol 532 |
Purity 96% |
100uL(2%) 250uL(5%) |
|
Protein solution (GFP-inJanus, BFP-inJanus, RFP-inJanus, RFP-inJanusm) |
BFP-inJanus(1.013mg/mL) GFP-inJanus(0.567mg/mL) RFP-inJanus(0.583mg/mL) RFP-inJanusm(0.475mg/mL) |
50uL protein solution+5950uL buffer |
Results of this experiment
Figure 14. The experiment used BFP-inJanus failed but other groups succeeded. We can see that 5%(v/v)of Berol 532 is suitable for all groups. After calculating, we found that the efficiency of systems adding 5% of Berol 532 are the highest. It’s correspond to the results measured by BCA.
Formal experiment using Berol 532 as detergent and HEPES(pH=7.3) as buffer.
We changed buffer and do this experiment again. Unfortunately, they all failed.
Formal experiment using Berol 532 as detergent and Reppal PES100 as polymer.
It has been reported that polymer can increase the efficiency of extraction and the system can be divided into two phases automatically without above cloud point. We used Reppal PES100 as polymer to do this experiment. However, it doesn’t work.
Formal experiment using Triton X-100 as detergent and HAc/NaAc as buffer.
The cloud point of Triton X-100 is 64-65 centigrade. We changed detergent and did this experiment again. We put the tubes in the heat block to make system separated. However, there aren’t any florescence in the detergent phase (the lower phase).
Formal experiment using Triton X-114 as detergent and HAc/NaAc as buffer.
The cloud point of Triton X-114 is 22 centigrade. We changed detergent and did this experiment again. We made the system separated at the room temperature, and used centrifuge to get a better separation. However, there aren’t any florescence in the detergent phase (the lower phase).
