Difference between revisions of "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 E4GI_core-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.