Figure 1.
Schematic diagram showing the construction of the prokaryotic expression vectors.
The hLSECtin-CRD (360bp) cDNA fragments were cloned into the prokaryotic expression vector pET28b-Tat, pET28b, pET22b (+) and pGEX-6P-1 to construct the prokaryotic expression vectors pET28b-Tat-hLSECtin-CRD, pET28b-hLSECtin-CRD, pET22b(+)-hLSECtin-CRD and pGEX-6P-1-hLSECtin-CRD.
Figure 2.
Identification of the protein expression level of Tat-hLSECtin-CRD and Tat-free hLSECtin-CRD at different time points by SDS-PAGE and Western blot with equal mass proteins, respectively.
a: SDS-PAGE analysis of the total protein, the supernatant fraction and the sediment fraction of transformed E. coli with vectors pET22b(+)-hLSECtin-CRD, pGEX-6P-1-hLSECtin-CRD; b, c: SDS-PAGE and corresponding Western blot analysis of the total protein, the supernatant fraction and the sediment fraction respectively using GAPDH as a loading control at different time points; d: Histograms constructed from the Western blotting data show the difference in expression levels between Tat-hLSECtin-CRD and Tat-free hLSECtin-CRD expression fractions at different induction time points. ‘Control (Null)’ indicate the empty vector pET28b. ‘Control (Tat)’ indicate the empty vector pET28b-Tat. The arrowheads indicate target proteins. **: p < 0.01, as compared to Tat-free hLSECtin-CRD.
Figure 3.
Identification of the subcellular microstructure of overexpressed Tat-hLSECtin-CRD or Tat-free hLSECtin-CRD bacterial cells by transmission electron microscopy.
The arrowheads indicate different aggregation level of Tat-hLSECtin-CRD and Tat-free hLSECtin-CRD. a: pET28b(control); b: pET28b-Tat-hLSECtin-CRD, the red arrows indicate the denser region of Tat-hLSECtin-CRD in cytosol ; c: pET28b-hLSECtin-CRD, the red arrows indicate the dot denser region of Tat-free hLSECtin-CRD in cytosol.
Figure 4.
The mannose-binding activity of Tat-hLSECtin-CRD using mannose-agarose columns.
The western blot showing the mannose-binding activity of washed and eluted fractions. A portion of Tat-hLSECtin-CRD proteins had bound on mannose-agarose column.