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INP-N ( N-terminus of Ice-nucleation protein ) from Pseudomonas syringae Sequence and Features

Ice-nucleation protein (INP) is a secreted outer membrane protein, which is widely distributed in Pseudomonas syringae, Pseudomonas fluorescens and other Gram-negative bacteria. Compared with other surface carrier proteins, ice-nucleation protein has the advantage of stably expressing heterogeneous proteins and displaying proteins with larger molecular weight. This part includes the sequence of INP-N (N terminus of Ice-nucleation protein), which is much shorter than INP and INP-NC yet still functions to anchor the target protein on the surface of the cell outer membrane. To allow this part fusion with other protein, we added an additional linker sequence at the 3'.

Usage and Biology

We employed this part to fuse the cellulases with INP-N to accomplish the cellulose degradation on the surface of bacteria. We linked the cellulases Cex and CenA respectively into the pET30a(+) backbone, which contains lacZ fragment so that the heterogeneous proteins can be induced by IPTG. The plasmids were transferred into BL21(DE3) strain and we induced these recombinants overnight under the condition of 16℃ 0.08 mM. The expression of fusion proteins was determined by SDS-PAGE (Figure 1).

Fig. 1 SDS-PAGE assay for fusion protein

To determine if the enzymes were anchored on the surface of the cell, we detected the presence of the fusion protein by immunofluorescence staining, then employed the undisrupted cells expressing fused enzymes to the enzyme activity assays.

Microscopy Observation

6His tag was added to present after the original cellulases (CenA and Cex) sequence and the fused cellulases (INP-CenA and INP-Cex) sequence as the antigen to be targeted by the primary antibody. Logically, since the Cellulase-6His is originally expressed in the interior of the cell, we would not detect the fluorescent signals in the sample of CenA and Cex, while the fluorescent signals are detectable for INP-Cellulase-6His due to the cell outer membrane anchoring effect. We observed the E.coli cells expressing the original proteins (CenA and Cex)and the fusion proteins (INP-CenA and INP-Cex)under the fluorescence microscopy`s 20X objective (Figure 2) and confocal fluorescence microscopy`s 100X objective (Figure 3).

Fig. 2 E.coli cell immunofluorescence staining observation via fluorescence_microscopy 20x objective

Fig. 3 E.coli cell immunofluorescence staining observation via confocal fluorescence microscopy, 100x objective (images are local zoomed)

Under the same condition of 20X magnification and 355ms for exposure, we noted that the fluorescent signals of the unfused cellulases field are dimmer than those of the fused cellulases field on average. To examine it more clearly, we observed the slices with the confocal fluorescence microscopy. The field of fusion protein samples showed that some foci are located on the borders of the cells, while this phenomenon was not observed in the field of the unfused protein samples. But due to the minuscule size of E.coli cells, our equipment falls short when trying to determine whether the fluorescent dot on a single cell is located on the outer membrane surface or not.

Fusion enzyme activity assay

The effect of fusion on enzyme activity was detected by measuring the cellulose degradation ability using CMC-Na as the substrate. We employed the procedures used by UESTC-China yet under the condition of citric acid-sodium citrate buffer with pH 4.8 and 50 ℃ for reaction temperature. We measured the cellulose degradation abilities of the supernatant of disrupted cell contents as well as the undisrupted cell suspensions. According to the standard curve of glucose concentration, we determined the activities of unfused enzymes CenA and Cex, and fusion enzymes INP-CenA and INP-Cex.(Figure 4)

Fig. 4 Enzyme activities assay (pH4.8 50℃)

From the data, we summarized that the cellulases` activities were not affected remarkably with the presence of INP-N. Also, the difference of enzyme activities between the ultrasonic-disrupted samples and undisrupted samples provided another evidence of the anchoring effect of INP-N. Since the fusion protein is anchored in the outer membrane surface, which would appear in the sediments after centrifugation，the samples of suspension with fused cellulases showed the relatively low level of the activity, compared with samples of unfused ones.