Fig 1.
Expression of mouse Chit1 in the cytoplasm of E. coli using pET system.
(A) Schematic representations of the E. coli-expressed mouse mature Chit1-V5-His. Mouse Chit1 is a secreted protein with a molecular mass of approximately 50 kDa, which contains an N-terminal catalytic domain and a C-terminal chitin-binding domain (CBD). (B) 10% SDS-PAGE of the total E. coli extract, soluble and insoluble recombinant proteins from E. coli. The proteins in the gel were visualized by CBB staining. (C) Western blot analysis of the recombinant proteins using anti-V5-antibody. Proteins were run by SDS-PAGE and transferred to a PVDF membrane. (D) Analysis of soluble and insoluble Chit1-V5-His. The recombinant proteins were separated from the soluble fraction by Ni Sepharose. In addition, insoluble-Chit1-V5-His was denatured by 8 M urea and refolded on Ni Sepharose. (E) Comparison of the chitinolytic properties of the purified recombinant proteins from soluble and insoluble fractions in 50 μL reactions using McIlvaine’s buffer (pH 5.0) at 37°C for 30 min as described in the Materials and Methods.
Fig 2.
Production of the Chit1 in the periplasmic space of E. coli as a fusion of Protein A-Chit1-V5-His.
(A) Schematic representation of the E. coli-expressed pre-Protein A-Chit1-V5-His. (B) 10% SDS-PAGE analysis of the recombinant proteins from the culture medium (Med), periplasmic fractions (Peri 1 and Peri 2), cytoplasmic soluble fraction (Cyto) and the insoluble fraction (Insol) from E. coli. The proteins were visualized by CBB staining. (C) Western blot analysis of the recombinant proteins using anti-V5 antibody. (D) Chromatogram of Protein A-Chit1-V5-His using Hitrap Q HP columns. Bound proteins were eluted with linear gradient of 0 to 1.0 M NaCl. First peak (bold lined) was pooled. (E) SDS-PAGE analysis of the protein fractions purified using the Ni Sepharose, followed by Hitrap Q HP columns. Purified protein were electrophoresed and visualized by staining with CBB. Western blot was performed with anti-V5-HRP antibody.
Table 1.
Subcellular distribution of chitinolytic activity of Protein A-Chit1-V5-His in E. coli.
Fig 3.
Comparison of the chitinolytic properties of mouse Chit1 prepared from E. coli and CHO cells.
(A) The schematic representations of the CHO-expressed pre-Chit1-V5-His. (B) pH profile of Chit1-V5-His. (C) We first measured the chitinolytic activity of the enzyme preparations from CHO cells and E. coli in a volume of 50 μL in McIlvaine's buffer (pH 5.0) at 37°C for 30 min. Then we adjusted the enzyme solutions to have same activity. We analyzed the immunoreactivities of these enzymes by Western blot using anti-V5 antibody. (D) The enzyme fractions with the same chitinase activities were visualized by Western blot using anti-V5 antibody. Molecular mass of Protein A-Chit1-V5-His expressed in E. coli was higher than that of Chit1-V5-His. CHO-expressed Chit1 and E. coli-produced Chit1 gave similar signals at approximately 52 kDa and 67 kDa, respectively.
Fig 4.
Characterization of the E. coli-expressed Chit1 activity.
(A) pH profile, (B) temperature profile, (C) pH stability profile and (D) affinity of Protein A-Chit1-V5-His to chitin beads. Chitinolytic activity (A~C) was measured as described in the Materials and Methods. The values represent percentage of the maximum activity obtained in each series of experiments. Error bars represent mean ± standard deviation from a single experiment conducted in triplicate. (D) Protein A-Chit1-V5-His and Protein A-V5-His were mixed and loaded onto chitin bead columns. Chitin binding assays were performed at pH 2.0, 5.0 and 7.0 as described in the Materials and Methods. The bound and unbound fractions were analyzed by Western blot using anti-V5 antibody.
Fig 5.
Degradation of colloidal and crystalline chitin and various GlcNAc oligomers by the recombinant Chit1.
Crystalline chitin (A), colloidal chitin (B), (GlcNAc)6 (C), (GlcNAc)5 (D), (GlcNAc)4 (E) and (GlcNAc)3 (F) were used as substrates in McIlvaine's buffer (pH 5.0). Reactions were conducted for 10 min, 1 h or 16 h at 37°C. Chitin fragments generated by the recombinant proteins were analyzed by fluorophore-assisted carbohydrate electrophoresis. Chitin oligomers are shown in the left margin as standards. Protein A-Chit1-V5-His released primarily (GlcNAc)2 fragments from chitin substrates.
Fig 6.
The pH-dependent chitin degradation by the recombinant Chit1.
Chit1 chitinase activity was investigated by incubating the enzyme with 4NP-(GlcNAc)2 (A), (GlcNAc)3, (B), colloidal chitin (C) or (GlcNAc)6 (D) as substrates at pH 2.0~8.0. Reactions were conducted for 30 min at 37°C, followed by labeling with a fluorophore and separated by PAGE as described in Materials and Methods. Chitin oligomers are shown in the left margin as standards.