Part:BBa_K2918028

Characterization of the SSB protein

For expressing our constructs we used PUREfrex system. This is an E.coli based cell-free protein synthesis system and it contains all the elements to make in vitro translation-transcription possible. A 10-μL reaction consists of 5 μL feeding buffer, 0.5 μL enzyme solution, 1 μL ribosome solution, 5 nM DNA and RNAse-free milliQ for filling up the volume. For fluorescent labeling, 0.5 μL of BODIPY-Lys-tRNA_Lys (FluoroTectTM GreenLys, Promega) was added, this binds to the translation products at the lysine residues sites.The proteins were identified by an 18% SDS-PAGE gel and mass spectrometry. From the 10-μL reaction, 8 μL was loaded on the SDS-PAGE while the other 2 μL was analysed by the mass spectrometer.

SDS-PAGE
After expressing the SSB protein for 3 hours, the sample was treated with RNAse (RNaseA Solution, Promega) for 30 minutes. To denaturate the protein the sample is also treated with 2x SDS loading buffer with 10 mM dithiotreitol (DTT) for 10 minutes at 90°C. Samples were loaded on a 18% SDS-PAGE (polyacrylamide gel electrophoresis) gel. Visualization was performed on a fluorescence gel imager (Typhoon, Amersham Biosciences) using a 488-nm laser and a band pass emission filter of 520 nm.

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  Figure 1: SDS-PAGE gels of DSB after protein purification. The gel features the ladder(L)

An SDS-PAGE was carried out for the SSB protein with 3 different promoter strengths: Wild-Type, 0.5 and 0.1. For a control PURE solution without any DNA was used. As can be concluded from the figure, in the sample containing the p5 protein a band can be found at the expected height(13kb). The band is also absent in the control, indicating that the p5 protein was successfully produced in the PURE system using this construct.

Mass Spectrometry

Next to the SDS-PAGE, mass spectrometry was used to confirm the identity of the proteins. To do this, a sequence unique to the SSB’s amino acid sequence were chosen and screened for their presence in the PURE system. For the p5 the peptide sequences are: IFNAQTGGGQSFK and TVAEAASDLIDLVTR. Data was normalized to the presence of the elongation factor EF-TU, which can be found in the same amount in all PURE system solutions.

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  Figure 2A: Identification in mass spectrometry of one peptide (IFNAQTGGGQSFK) of p6 sample after purification
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  Figure 2B: Identification in mass spectrometry of one peptide (TVAEAASDLIDLVTR) of p6 sample after purification

The intensity of the mass spectrographs shown in Figure 2 only reflect the occurrence of a given sequence in the sample. The mass spectrometer looks for the peptide sequences that is selected. These peptide sequences were only present in the samples that were expected. The difference in height can be attributed to the strength of the promoters, less peptides were measured with decreasing strength. In conclusion, the results were positive and the identity of the proteins could be verified by mass spectrometry.

In Vitro replication