Fig 1.
Schematic illustration of disrupted triple helix digestion by trypsin and TEV protease.
(A) An intact triple helix is resistant to digestion by trypsin. (B) Due to mutations and natural interruptions, the triple helix become susceptible to trypsin digestion, which might result in protein degradation. In contrast, TEV protease does not degrade disrupted triple helix, enabling the isolation of mutant triple helices for more precise characterization and functional studies and for use as a potential biomaterial.
Fig 2.
Schematic illustration of the design of recombinant bacterial collagens.
(A) VCL2 with a protease cleavage site (highlighted by “↑”) and an IBS within each CL domain. (B) Version of the VCL2 construct with Gly→Arg within the IBS, where the second Gly residue of GFPGER was mutated to Arg (highlighted by “★”). The ‘CPPC’ domains represent the amino acid sequences inserted at the N- and C- terminal of the CL domains to aid in stability. (C) Description of the designed protein constructs.
Fig 3.
Protein expression of VCL2 was optimized to achieve high expression and protein solubility.
(A) SDS-PAGE of the VCL2 purified proteins expressed in E. coli BL21 and BL21-DE3. Protein expression in BL21-DE3 cells resulted in purification of VCL2 with high purity, without any degradation products. (B) SDS-PAGE of insoluble fractions (IF) and soluble fractions (SF) of VCL2 after induction with varying IPTG concentrations, where lower IPTG concentrations induced high protein expression. Molecular Weight standards are in kDa. Conc. = Concentration.
Fig 4.
Ni-NTA purification of expressed proteins lead to high purity.
SDS-PAGE of the purified proteins, (A) VCL2-Trypsin, (B) VCL2-TEV, (C) VCL2(G→R)-Trypsin, and (D) VCL2(G→R)-TEV. Cell lysate was incubated with Ni-NTA resin and FT was collected. The column was washed with binding buffer, high salt buffer, and low imidazole buffer to remove non-specific bound proteins. His-tagged protein was eluted with buffer containing 100 mM (Elution-I), 250 mM (Elution-II), and 500 mM (Elution-III) imidazole with three fractions collected per concentration. Molecular weight standards in kDa, Ly = lysate, and FT = Flowthrough (containing the unbound protein).
Fig 5.
Incubation of distorted triple helices with trypsin demonstrated protein digestion while incubation with TEV protease allowed isolation of CL domains containing Gly→X mutations.
SDS-PAGE of (A) VCL2-Trypsin, (B) VCL2(G→R)-Trypsin, (C)VCL2-TEV, (D) VCL2(G→R)-TEV, and (E) VCL2(G→R)-TEV purified proteins digested with either trypsin or TEV protease for different time points (0 mins, 2 mins, 15 mins, and O/N). Digestion of VCL2 with trypsin and TEV protease resulted in removal of His6-tag and V-domain and isolation of CL2. However, digestion of VCL2(G→R) with trypsin led to protein digestion starting at 2 mins only. Digestion of VCL2(G→R)-TEV with TEV protease resulted in the isolation of CL2(G→R) protein without degrading the protein. Molecular weight standards in kDa, O/N = Overnight digestion with the protease.