Figure 1.
Primary sequence analysis of four SSB proteins.
The amino-acid sequences of TTH, BL21, KOD and SSOB SSB proteins (NCBI access number: AP008226, AM946981, GM017008, and NP_343725, respectively) were aligned using a multiple alignment program for amino acid or nucleotide sequences (http://mafft.cbrc.jp/alignment/server/). The structure of the DNA-binding domain was marked according to reference 3. The conserved DNA-binding sites (amino acids labelled in red) were identified by the NCBI CD-Search & Batch CD-Search online programs. One dot represents a semi-conserved amino acid change, two dots represent a conserved amino acid change, and stars represent identical amino acids. All SSB proteins were abbreviated by the nomenclature of their ancestor bacteria.
Table 1.
Summary of SSB proteins properties.
Figure 2.
SDS-PAGE and native PAGE analysis of SSB proteins.
A. Purity and protein molecular weight were visualised by SDS-PAGE in a 12.5% polyacrylamide gel containing 0.1% SDS under reducing conditions. B. Functional SSB protein polymers were notarised by 3.5–5–12% non-denaturing gel electrophoresis.
Figure 3.
Non-denaturing gel electrophoresis (3.5–5–12%) was used to confirm SSB protein binding to ssDNA. Relative dsDNA was used as a binding control. M, monomer; D, dimer; T, tetramer. A, ssob binding ssDNA analysis. B, tth binding ssDNA analysis. C, kod binding ssDNA analysis. D, bl21 binding ssDNA analysis.
Figure 4.
Heat-resistance analysis of four SSB proteins.
Four SSB proteins were heated at 95°C for 1, 2, 3, 4, 5, 120, 240, and 600 min, respectively, and their binding activities were assessed by native PAGE. Black arrow indicates the SSB protein–ssDNA binding complex. Non-heat-treated SSB proteins were used as controls.
Figure 5.
Effect of SSB proteins on long-fragment human genome amplification.
Human beta globin gene fragments (5, 9, and 13 kb) were used as templates to assess the effect of SSB proteins on PCR amplification under optimised conditions.
Figure 6.
TAE agarose gel electrophoresis (1.2%) and an in vitro–transcribed HCV RNA were used to assess SSB protein-binding activity. Non-denatured RNA and denatured RNA were mixed with 240 and 600 min heat-treated SSB proteins. Zero minutes represents SSB proteins that were not heated, and nude RNA (denatured at 65°C for 5 min) represents RNA alone.
Figure 7.
HCV RNA was denatured by incubation at 65°C for 5 min. The threshold and a 10-fold higher Benzonase doses were added to the SSB protein–HCV RNA mixture. HCV RNA with no SSB proteins was used as the control.
Figure 8.
SSB proteins modulated HCV RNA metabolism.
A. In vitro–transcribed HCV RNA (500 ng) was transfected into 1×106 Huh7 cells with or without 10 or 20 ng of SSB proteins. The BSA was used as random protein control. After the 72-h incubation, the cells were harvested for RNA expression analysis. A HCV-infected patient serum was used as the primary antibody to probe HCV proteins expressed by transfected HCV RNA. The positive cells were visualised using a FITC-labelled goat anti-human antibody followed by FACS analysis. All p values marked by star are less than 0.05. B. The mean fluorescence intensity of the HCV protein positive cells were calculated by FACS. All p values marked by star are less than 0.05. C. cellular localization and co-localization analysis, the above cells were double stained with anti-HCV serum and anti-His (all SSB proteins were labelled with His-tag) antibody, localization of SSB proteins and co-localization of SSB and HCV protein were evaluated by a cofocal microscope.