Part:BBa_K1582000
inJanus from Grifola frondosa
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 121
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.
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