Production of recombinant human procollagen type I C-terminal propeptide and establishment of a sandwich ELISA for quantification

Abstract

Procollagen type I carboxy-terminal propeptide (PICP), derived from type I procollagen, has been identified as an indicator of type I collagen synthesis in bone matrix formation and skin recovery. PICP is a heterotrimeric glycoprotein consisting of two α1 chains (PICPα1) and one α2 chain (PICPα2). Here, we report the recombinant expression of human PICP using a mammalian expression system. Co-expression of PICPα1 and PICPα2 in HEK293F cells resulted in the production of functional PICP in the correctly assembled heterotrimeric form. Using the recombinant PICP as an antigen, we isolated PICP-specific human monoclonal antibodies from phage-displayed antibody libraries and raised rabbit polyclonal antibodies. Using those antibodies, we then developed a sandwich ELISA for PICP with a limit of detection of 1 ng/mL and a measurable range of 1-640 ng/mL. Both intra- and inter-assay imprecision values were <10%. For measuring PICP levels in human fibroblast cellular extracts and culture supernatants and a human serum, the developed ELISA kit displayed comparable performance to that of a commercialized kit. Our results provide an efficient production strategy for recombinant PICP, facilitating the generation of PICP-specific antibodies and development of PICP sandwich ELISA, with potential use in clinical diagnosis of serum samples and testing of cosmeceutical ingredients in fibroblast cell cultures.

Figure 1

Design and expression of recombinant human PICP protein. (a) Schematic diagram showing the generation of PICP from type I procollagen by enzymatic cleavage (the red arrow indicates the cleavage site). PICPα1 and PICPα2 chains are represented as strings in cyan and magenta colors, respectively. Disulfide bonds are indicated with yellow lines. Each end of the strings is labeled with the residue number of type I procollagen. (b) Design of mammalian expression plasmids to secrete PICPα1, PICPα2, and PIIICPα1. The 8× His tag (green) and c-myc tag (purple) are indicated with colored rectangles. (c) Schematic diagram showing recombinant expression of the correctly assembled PICP and PIIICP. Two PICPα1 chains (cyan) and one PICPα2 chain (magenta) assemble into heterotrimeric PICP (upper), while three PIIICPα1 chains (light yellow) assemble into homotrimeric PIIICP (lower). (d) Western blot analysis of purified PICP and PIIICP. The proteins were subjected to SDS-PAGE in non-reducing or reducing conditions, and the gel was blotted with anti-His antibody (left) and anti-c-myc antibody (right). The corresponding full-length blots are shown in Supplementary Fig. 4.

Figure 2

SDS-PAGE and SDS-CGE analyses of purified recombinant PICP and PIIICP. (a) Non-reducing and reducing SDS-PAGE analyses of the purified recombinant PICP and PIIICP. The arrows dictate the eluted position of indicated proteins. The number below the band indicates the relative value of band intensity of each band to that of PICP heterotrimer band in the non-reducing conditions. (b) Gel-like image of SDS-CGE analysis of the purified recombinant PICP and PIIICP using a Protein 230 assay kit. In non-reducing conditions, PICP and PIIICP appeared mainly as a single band at the expected size. In reducing conditions, PICP and PIIICP showed two bands for PICPα1 and PICPα2 and a single band for PIIICPα1, respectively. (c) Electropherogram of SDS-CGE analysis of the recombinant PICP and PIIICP shown in (b). Significant peaks are marked with black arrow heads, with the calculated molecular weights. The inset table below shows the precise quantity and ratio of each peak area in the electropherogram. The raw images of SDS-CGS analysis are shown in Supplementary Fig. 5.

Figure 3

Isolation and characterization of anti-PICP 2D and 4G mAbs. (a) Phage ELISA of isolated individual phage to determine the binding activity of plate-coated PICP (top), PIIICP (middle), and PICP with its soluble competitor PIIICP (bottom). Phages were isolated after 4 rounds of panning of human scFv phage library against recombinant PICP in the presence of competitor PIIICP. (b) Indirect ELISA of 2D and 4 G clones in the format of scFv (0~2 μg/mL) and IgG1 (0~1 μg/mL) to determine their binding specificity to plate-coated recombinant PICP (left) and recombinant PIIICP (right). (c) Representative SPR sensorgrams showing the kinetic interactions of IgG1-formatted 2D and 4 G mAbs with recombinant PICP. The inset table shows the kinetic interaction parameters. Each value represents the mean ± SD of five data sets.

Figure 4

Specific binding of 2D mAb to native PICP. (a) Western blot analysis of the anti-PICP 2D mAb immunoprecipitation (IP) for culture supernatants of primary dermal fibroblast cells. The corresponding full-length blots are shown in Supplementary Fig. 6. (b) Identification of PICP protein in the immunoprecipitated gel band by LC/MS. The three different peptides and one peptide identified from the PICPα1 and PICPα2 chains, respectively, were indicated in distinct colors in the amino acid sequence. The left panels show the representative MS/MS spectrum for the identified peptides of DRDLEVDTTLK (652.83 m/z, +2) from PICPα1 (top) and EMATQLAFMR (615.28 m/z, +2) from PICPα2 (bottom), underlined in each sequence.

Figure 5

Establishment and validation of a sandwich ELISA for PICP quantification. (a) Schematic representation of the developed sandwich ELISA (see text for details). (b) Standard curve of the developed sandwich ELISA for quantification of recombinant human PICP. The data was fitted into a 4PL model to assess the correlationship between absorbance and the increasing concentration of recombinant PICP (r ² = 0.99). (c) Western blotting to monitor the levels of PICP, type 1 procollagen, and phosphorylated Smad2 in LX-2 cells. For western blot analysis, an equal quantity of LX-2 cellular lysate was loaded using β-actin as a loading control. The number below the panel indicates the relative value of band intensity of the proteins compared with that of control after normalization of the band intensity to that of β-actin for each sample. The corresponding full-length blots are shown in Supplementary Fig. 7. (d) Quantification of PICP levels in the cellular extracts and culture supernatants of TGF-β stimulated LX-2 cells using the developed sandwich ELISA and the commercially available Takara ELISA kit (MK101). Error bars, ±SD (n = 3). (c,d) LX-2 cells were treated with medium (control) or the indicated concentrations of TGF-β for 12 h, and then the soluble cellular extracts and culture supernatants were subjected to the analyses. (e) Measurements of PICP levels in a normal human serum diluted at the indicated rate using the developed sandwich ELISA kit and the commercially available Takara ELISA kit (MK101). Error bars, ±SD (n = 3). Inset shows the equation for the fitted line and the correlation coefficient value (r ²).