Difference between revisions of "Part:BBa K2933285"

Latest revision as of 13:21, 21 October 2019

smURFP（mutation Y56R） + Linker A + RBS I + HO-1 I

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Sequence and Features

Usage and biology

smURFP (small ultra-red FP) is an important part in our group. It is desirable for our BV detection and in-vivo imaging because with it molecule less light is scattered, absorbed, or re-emitted by endogenous biomolecules compared with cyan, green, yellow and orange FPs. smURFP can covalently attaches a biliverdin(BV) chromophore without a lyase, and has 642/670 nm excitation - emission peaks, a large extinction coefficient and quantum yield, and photostability comparable to that of eGFP.
In order to fluorescence, Site-directed mutation smURFP must be combined with biliverdin (BV) .So we construct the surface display system to make in-vivo imaging come true. To construct the surface display system, the gene of fluorescent protein---smURFP and the gene of the anchoring protein should be connected to the same expression vector. After the recombinant plasmid is transferred to the target bacteria, the fluorescent protein and anchoring protein will express at the same time and become fusion protein, and then the fluorescent protein will be carried to the cell surface by anchoring protein. With the added biliverdin, fluorescent protein will combine with biliverdin and glow on the cell surface. For more information, see the part BBa_K2328027.

In this part, we mutated the tyrosine of smURFP at position 56 to arginine.This change led to a significant increase in smURFP's ability to bind to the small molecule BV.
We co-expressed smURFP and Biliverdin-producing protein heme oxidase (Heme Oxygenase-1, HO-1) in E. coli and then purified the smURFP protein that binds BV small molecules. We used wild-type smURFP as a control and measured the absorbance at specific wavelengths of proteins, small molecule BV, and smURFP-BV complexes by ultraviolet spectrophotometer (280 nm, 388 nm, 642 nm, respectively). We normalized the wild type and mutant absorbance at 280 nm. Through data analysis, we found that the ratio of small molecule BV binding to protein in the mutant complex was significantly greater than that of wild type, which significantly demonstrated that the ability of the mutant to bind small molecules BV had been greatly improved.

Molecular cloning

We used the vector pet-28b-sumo vector to construct our expression plasmid. We converted the plasmid to E. coli BL21(DE3) for expression and purification.

Figure 1. (a)Cloning of wild type smURFP gene and ho-1 gene. (b)Overlap PCR ligation. (c)Enzyme digestion validation.

Figure 2. (a)Cloning of mutation smURFP(Y56R) gene. (b)Overlap PCR ligation.(c)Enzyme digestion validation.

Protein expression and purification

Pre-expression: The bacteria were cultured in 5mL LB liquid medium with ampicillin(100 μg/mL final concentration) in 37℃ overnight.
Massive expressing: After taking samples, we transfered them into 1L LB medium and add antibiotic to 100 μg/mL final concentration. Grow them up in 37°C shaking incubator. Grow until an OD 600 nm of 0.8 to 1.2 (roughly 3-4 hours). Induce the culture to express protein by adding 1 mM IPTG (isopropylthiogalactoside, MW 238 g/mol). Put the liter flasks in 16°C shaking incubator for 16h.
Affinity Chromatography:
We used the Ni Agarose to purify the target protein. The Ni Agarose can combine specifically with the Ni-Sumo tag fused with target protein.

• First, wash the column with water for 10 minutes. Change to Ni-binding buffer for another 10 minutes and balance the Ni column.
  
• Second, add the protein solution to the column, let it flow naturally and bind to the column.
  
• Third, add Ni-Washing buffer several times and let it flow. Take 5ul of wash solution and test with Coomassie Brilliant Blue. Stop washing when it doesn’t turn blue.
  
• Forth, add Ni-Elution buffer several times. Check as above.
  
• Fifth, collect the eluted proteins for further operation.
  

Gel filtration chromatography:
The collected protein samples are concentrated in a 10 KD concentrating tube at a speed of 3400 rpm and concentrated for a certain time until the sample volume is 500 μl. At the same time, the superdex 75 column is equilibrated with a buffer to balance 1.2 column volumes. The sample is then loaded and 1.5 cylinders are eluted isocratically with buffer. Determine the state of protein aggregation based on the peak position and collect protein samples based on the results of running the gel.

Figure 3. (a)Separation of peak position from molecular sieves of smURFP and BV complexes.(b)SDS-PAGE

Figure 4. (a)Separation of peak position from molecular sieves of smURFP and BV complexes.(b)SDS-PAGE

Improvement of the ability to binding BV

We used wild-type smURFP as a control and measured the absorbance at specific wavelengths of proteins, small molecule BV, and smURFP-BV complexes by ultraviolet spectrophotometer (280 nm, 388 nm, 642 nm, respectively). We normalized the wild type and mutant absorbance at 280 nm. Through data analysis, we found that the ratio of small molecule BV binding to protein in the mutant complex was significantly greater than that of wild type, which significantly demonstrated that the ability of the mutant to bind small molecules BV had been greatly improved.

Figure 5. smURFP, small molecule BV and BV- smURFP complexes were quantified by measuring the absorption values of wavelengths of 280 nm, 388 nm and 642 nm, respectively. (a)Absorption values of the wild type smURFP-BV complexes solution. (b)Comparison of absorption values of the wild type smURFP-BV complexes and the mutation smURFP(Y56R)-BV complexes.

Reference

[1] Rodriguez EA,Tran GN , Gross LA, et al. A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein .[J].NATURE METHODS,2016:763-769.