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smURFP (I, codon-optimized for Escherichia coli) (without terminator codon TAA)

Sequence and Features

Usage

Biology

Reference

Results

We did many experiments on smURFP.

Protein production and purification

After the expression of protein by E.coli BL21, the products were purified for several times. Figure 1 reflects the condition of sample after purification of the nickel column. The first is before purification, and 2 and 3 were purified ones.

Figure 1. To test the purification efficiency and condition of Nickel Column Purification is via SDS-PAGE method. (a) The marker. (b) The 20 μl sample of E.coli crushing fluid before Nickel Column Purification with the target part of smURFP and his-sumo tag, around 26 kD. (c) The 20 μl sample of the solution with the target part is after Nickel Column Purification. (d)The 20 μl sample of the solution after is Nickel Column Purification is before the sumo protease digestion, with sumo protease around 13 kD. (e) The 20 μl sample of the Nickel medium is after 12 h sumo protease digestion with the target protein smURFP, around 12 kD. (f) The 20 μl sample of the eluent is with the protein smURFP. (g) The 20 μl sample of the Nickel medium after the elution to test whether the target protein is all in the solution.

We then used the AKTA system and molecular sieve system for further purification.

Figure 2. Result of purification of AKTA system.

Figure 3. Result of purification of molecular sieve system.

Figure 4. . To test the purification efficiency and condition of AKTA Ion Exchange Chromatography and Molecular Sieve Purification. (a) The 20 μl sample of the solution before Ion Exchange Chromatography Purification is as a compare. (b)~(h) The 20 μl sample of the solution is after Ion Exchange Chromatography Purification. (i) The marker. (j) The 20 μl sample of the solution before Molecular Sieve Purification is as a compare. (l)~(o) The 20 μl sample of the solution is after Molecular Sieve Purification.

Protein crystallization

Table 1. Result of proper condition for crystallization.

Protein and BV

We test the fluorescence changes of different concentration of smURFP and BV and the fluorescence.

Figure 5. A series of curves of fluorescence intensity changes of different concentration of smURFP and BV.

Co-expression with HO-1

Plasmid pET28b with smURFP and HO-1 gene were transformed into E. coli BL-21. We used this induced bacteria to confirm the fluorescence and data showed a relatively high value, as shown in table 1.

Table 1. Result of Microplate Reader in the black 96-well plate. Tube 1 and 2 are experimental group, and tube 3 is the control group.

Then laser confocal microscopy was use to observe these bacteria, activate light of 640nm was used, as shown in Figure 2.

Figure 2. The result after induction, the upper one is the control group, and the inferior one is the experimental group.