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.
Highlights
Collagens are the major structural proteins of the extracellular matrix that play a crucial role in providing connective tissue structural integrity and homeostasis[1]
By quantifying the relative band intensity on SDS-PAGE gel and the peak area of SDS-capillary gel electrophoresis (SDS-CGE) electropherogram of the proteins, which correspond to the amount of protein in the band, we analyzed the ratio of the two separated bands observed in the procollagen type I carboxy-terminal propeptide (PICP) lane under reducing conditions. Both band intensity and peak area of the two bands represented a ratio of approximately 7:3 (Fig. 2a,c and Supplementary Fig. 3), which is almost the same composition ratio of natural heterotrimeric PICP (PICPα1 chain: PICPα2 chain = 2:1 molar ratio)[10,13]. These results suggest that recombinant PICP was dominantly expressed in the correctly assembled heterotrimeric molecule composed of two PICPα1 chains and one PICPα2 chain, like in naturally occurring PICP10,13
These results strongly suggest that the isolated 2D monoclonal antibodies (mAbs) is specific to natural PICP, and the recombinant PICP can be used as a high-quality antigen to screen anti-PICP antibodies as a substitute to PICP purified from human fibroblast cell cultures
Summary
Collagens are the major structural proteins of the extracellular matrix that play a crucial role in providing connective tissue structural integrity and homeostasis[1]. They are highly associated with bone metabolism, wound healing, and skin recovery, and their synthesis is tightly controlled and regulated[2]. There are approximately 28 different types of collagens, all of which are composed of three polypeptide chains (α chains) forming either homotrimers with three identical α chains or heterotrimers with two/three different α chains[1]. With unstable PINP in the serum, PICP serum levels are stoichiometrically related to the amount of type I collagen synthesized in the bone matrix[3,7].
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