Preparation and Characterization of Hydroxylated Recombinant Collagen by Incorporating Proline and Hydroxyproline in Proline-Deficient Escherichia coli

Abstract

Collagen possesses distinctive chemical properties and biological functions due to its unique triple helix structure. However, recombinant collagen expressed in Escherichia coli without post-translational modifications such as hydroxylation lacks full function since hydroxylation is considered to be critical to the stability of the collagen triple-helix at body temperature. Here, a proline-deficient E. coli strain was constructed and employed to prepare hydroxylated recombinant collagens by incorporating proline (Pro) and hydroxyproline (Hyp) from the culture medium. By controlling the ratio of Pro to Hyp in the culture medium, collagen with different degrees of hydroxylation (0-88%) can be obtained. When the ratio of Pro and Hyp was adjusted to 12:8 mM, the proline hydroxylation rate of recombinant human collagen (rhCol, 55 kDa) ranged from 40-50%, which was also the degree of natural collagen. After proline hydroxylation, both the thermal stability and cell binding of rhCol were significantly enhanced. Notably, when the hydroxylation rate approached that of native human collagen (40-50%), the improvements were most pronounced. Moreover, the cell binding of rhCol with a hydroxylation rate of 43% increased by 29%, and the melting temperature (Tm) rose by 5 °C compared to the non-hydroxylated rhCol. The system achieved a yield of 1.186 g/L of rhCol by batch-fed in a 7 L fermenter. This innovative technology is expected to drive the development and application of collagen-related biomaterials with significant application value in the fields of tissue engineering, regenerative medicine, and biopharmaceuticals.

Keywords: co-expression; hydroxylation rate; incorporation; recombinant collagen; triple helix.

Figure 1

Schematic diagram of preparation for hydroxylated recombinant collagen by incorporating Pro and Hyp.

Figure 2

Construction and verification of engineering bacteria. (A) Schematic of CRISPR-Cas9 gene editing technology for gene knockout. (B) Growth curve of E. coli MG1655 △proC in M9 basic medium. The black line refers to the addition of 120 mg/L of proline to the M9 basic medium. (C) Agarose gel electrophoresis analysis for gene knockout verification of proline-deficient strains. M: marker, Lanes 1, 4, 7, 10: verification of proC gene knockout; Lanes 2, 5, 8, 11: verification of ompT gene knockout; Lanes 3, 6, 9, 12: verification of lon gene knockout. (D) SDS-PAGE analysis of protein rhCol expression in different strains. M: protein marker, W: protein in whole cell solution, S: protein in supernatant, P: protein in precipitation. The red arrow indicates the location of the target protein. (E) Relative expression of the protein rhCol in different strains. (Each test with three replicates (n = 3). Data were analyzed by a one-way ANOVA using SPSS (version 22), and the results are presented as mean ± SD, *** p < 0.001).

Figure 3

Preparation of hydroxylated recombinant collagen by incorporation of Pro and Hyp from culture medium. (A) Flow chart of the novel system for preparation of hydroxylated recombinant collagen. (B) SDS-PAGE analysis of protein expression with different concentrations of Hyp incorporated. M: protein marker. The red arrow indicates the location of the target protein, and the red box highlights the most appropriate Hyp concentration. (C) SDS-PAGE analysis of protein expression with different concentrations of NaCl in M9 basic medium. M: protein marker. The red arrow indicates the location of the target protein, and the red box highlights the most appropriate Nacl concentration. (D) The effect of different OD₆₀₀ in LB medium before centrifugation on hydroxylation of recombinant collagen. In the culture medium, the concentrations of Pro and Hyp were 4 mM and 16 mM, respectively. The culture time after induction was 4 h. (E) The effect of different culture times after induction in M9 on hydroxylation of recombinant collagen. In the culture medium, the concentrations of Pro and Hyp were 4 mM and 16 mM, respectively. The OD₆₀₀ before centrifugation was 3. (F) The effect of different ratios of Pro to Hyp on hydroxylation of recombinant collagen (each test was performed with three replicates (n = 3). Data were analyzed by a one-way ANOVA using SPSS, and the results are presented as mean ± SD, ns > 0.05, 0.001 < ** p < 0.01, *** p < 0.001).

Figure 4

Structural analysis of rhCol. (A) Identification of hydroxylation sites in rhCol by LCMS/MS. “P” marked in red refers to hydroxylated proline; the gray part refers to the sequence that failed to be detected; “Inc” refers to rhCol prepared by incorporation; “P4H” refers to rhCol hydroxylated by proline hydroxylase BaP4H. (B) CD spectra of rhCol with different hydroxylation rates and different hydroxylation methods. The hydroxylation resulted in an obvious triple helix positive absorption peak at 221 nm. (C) CD thermal melting analysis of different rhCols. (D) SEM analysis of unhydroxylated rhCol. (E) SEM analysis of rhCol with a 43% hydroxylation rate. (F) SEM analysis of rhCol with an 88% hydroxylation rate.

Figure 5

Biological characterization of different rhCol samples. (A) Cell binding analysis of rhCol with different hydroxylation rates. These rhCol samples were prepared through the incorporation method. (B) Cell binding analysis of three collagens with nearly the same hydroxylation rate of around 43%, prepared through the incorporation method, rhCol hydroxylated by BaP4H, standard bovine type I collagen. (C) Cell viability analysis of rhCol with different hydroxylation rates. These rhCol samples were prepared through the incorporation method. (D) Cell viability analysis of three collagens with nearly the same hydroxylation rate of around 43%, prepared by incorporation method, rhCol hydroxylated by BaP4H, standard bovine type I collagen. Each test with three replicates (n = 3). Data were analyzed by a one-way ANOVA using SPSS, and the results are presented as mean ± SD, ns > 0.05, 0.01 < * p < 0.05, 0.001 < ** p < 0.01, *** p < 0.001.

Figure 6

The fed-batch fermentation of hydroxylated recombinant collagen by incorporation in a 7 L fermenter. (A) Cell growth curve (blue) and production of hydroxylated rhCol (red) in a 7 L fermenter. Arrows represent the transition point for protein induction and cultivation stage, L-Arabinose: the time of inducing T7RNA polymerase expression; medium exchange: the point of replacing fermentation medium with M9 fermentation medium; IPTG/Pro/Hyp: the time of inducing rhCol expression. (B) SDS-PAGE analysis of the production of hydroxylated rhCol during fermentation in a 7 L fermenter. M: protein marker.