Difference between revisions of "Part:BBa K1582000"

Revision as of 16:36, 23 September 2015

inJanus from Grifola frondosa

Sequence and Features

Usage

Janus is a kind of amphipathic protein which could self-assembly spontaneously. Due to its special properties, we could make many new applications. We use them as substrate 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. And 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.

Biology

Janus could be produced by filamentous fungi, such as Ascomycetes and Basidiomycetes, and their scientific name is hydrophobin. 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. One of the most important features of Janus is that they are able to assemble spontaneously into amphipathic monolayers at hydrophobic–hydrophilic interfaces.

There are two classes of Janus, which are divided by the stability of their self-assembly. inJanus from Grifola frondosa belongs to Class I. They could generate very insoluble assemblies, which can only be dissolved in strong acids such as trifluoroacetic acid or formic acid.

Protein Expression

This year, Nankai helped us to express inJanus in pichia pastoris.
To express inJanus in pichia pastoris, Nankai chose fed-batch fementation carried out in bioreactor with 1L initial volume. When the cell fresh weight reached to 200g/L, Nankai started inducement for 96h. What’s more, there is a higher production when dissolved oxygen was maintained at 15%-25% by turning up the methanol speed.
Follow is the figure shows the SDS-PAGE and western blot analysis of inJanus. And the inJanus molecule weight was 8KDa. In addition, inJanus in the broth was mainly monomer and dimer. The latter binds better with the primary antibody of Janus.

Figure 1. (a)silver staining along with SDS-PAGE and (b)western blot analysis of inJanus fermentation broth(lane 2,3), lane 1 was pure inJanus as control

To measure the yield of inJanus, Nankai measured the concentration of inJanus by UPLC. And Nankai used standard sample of sJanus of different concentration (0.1, 0.05, 0.025 mg/mL) to get a standard curve of concentration with adsorption area of UPLC.

Figure 2.(a) silver staining along with SDS-PAGE of Janus fermentation broth of different induction time(lane 1-4 24,48,72,96 h).(b)inJanus production and cell fresh weight in different time.

Figure 3.HPLC of pure inJanus HPLC

Super Protein Chip

Results

1. The optimal concentration of inJanus is 200μg/ml.
2. The optimal concentration of antigens (CEA, AFP and CA15-3) is 2.5μg/ml.
3. The optimal concentration of antibodies is 5μg/ml.

Figure 4.Hydrophobins sJanus show good effects of antibodies fixing. Here we use antibodies with green fluorescence tag as example. The spots with strong fluorescence represent successful binding between capture antibody (probe) and antigen (tumor marker).

Figure 5.Hydrophobins inJanus tend to show weak fluorescence signal. After compared experiments, we discovered that increasing the incubation time helps to improving detecting sensitivity.

Project Achievements
1.We introduced a new method of protein chip substrate modification by using Janus, and verified its feasibility.
2.We confirmed the optimal concentration of antigens and antibodies in the detection of tumor markers, which helps to save materials to the most extent and keep the reliability of the results at the same time.
3.The experiments were conducted with different kinds of antigens and antibodies, ensuring the universality of the tumor detection chip under a certain range.

Optimization

1.In the beginning, due to our inexperience, the concentration of antigens and antibodies was too high, which leads to excessive strong fluorescence signal intensity.
At first we thought it was caused by the improperly operation of microscope, but after literature reviewing and analyzing, we tried lowering the concentration about four times and achieved good results.

Figure 6.Here is the excessive strong fluorescence signal intensity caused by high concentration.

Figure 7.Good results were achieved after lowering the concentration.

2. In mid-stage, many crystallized structures appeared in the protein spot, and hence influenced the image quality and observation results. To solve this problem, we consulted large quantities of data and information, and discovered a similar case arising when solutions of antibodies and antigens were stored for too long, leading to degeneration. So we reformulated all the solutions we need, and crystallization disappeared. From this we learned that reagents should be used right after they were ready. In this case, another possible cause was that we used TBST in the last washing step, which may also lead to crystallization. In the upper course of experiments we used water instead of TBST. 

Figure 8.Crystallized structures appeared.

Figure 9.Crystallized structures disappeared after reformulating all the solutions we need.