Part:BBa_K1582004

GFP+sJanus Fusion Protein

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 976
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 644
  Illegal SapI site found at 985
  

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.

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. 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.