Production of Recombinant Human Type I Procollagen Trimers Using a Four-gene Expression System in the Yeast Saccharomyces cerevisiae

Abstract

The expression of stable recombinant human collagen requires an expression system capable of post-translational modifications and assembly of the procollagen polypeptides. Two genes were expressed in the yeast Saccharomyces cerevisiae to produce both propeptide chains that constitute human type I procollagen. Two additional genes were expressed coding for the subunits of prolyl hydroxylase, an enzyme that post-translationally modifies procollagen and that confers heat (thermal) stability to the triple helical conformation of the collagen molecule. Type I procollagen was produced as a stable heterotrimeric helix similar to type I procollagen produced in tissue culture. A key requirement for glutamate was identified as a medium supplement to obtain high expression levels of type I procollagen as heatstable heterotrimers in Saccharomyces. Expression of these four genes was sufficient for correct assembly and processing of type I procollagen in a eucaryotic system that does not produce collagen.Collagen is the single most abundant protein found in animals. It is found in all animals, including sponges. It is not expressed in yeast. In mammals, it is expressed in most tissues and plays both a structural as well as a signaling role in the development, maintenance, and repair of tissues and organs. More than 30 gene products compose the collagen family of molecules (1). Procollagens have several features and require numerous steps for production of functional molecules, including post-translational modifications (2). Key features in the collagen family are the formation of a triple helix composed of three polypeptide chains and the post-translational modification of proline residues to hydroxyproline, which provides stability of the triple helix against thermal denaturation and unfolding (T m ) 1 at the animal's body temperature (3). The content of proline and hydroxyproline is correlated with the temperature of an animal's environment (4). The triple helical domain of procollagen consists of -(GXY) n -repeats, where X and/or Y is frequently proline or hydroxyproline in the mature molecule. Prolyl 4-hydroxylase, an ␣ 2 ␤ 2 tetrameric enzyme composed of the prolyl hydroxylase ␣-subunit (␣PH) and the protein-disulfide isomerase (PDI) subunit in higher eucaryotes, is the enzyme that modifies proline residues to hydroxyproline. Additional steps for procollagen production include carbohydrate attachment, folding into a triple helix, secretion into the extracellular matrix, and cleavage by specific proteases to remove the propeptide domains to form mature collagen helices. A C-terminal non-helical propeptide facilitates the assembly of trimeric collagen molecules, leading to helix formation (5); the N-terminal propeptide may limit fiber diameter (6). The association and folding steps of three polypeptide chains that compose the triple helix potentially require chaperone functions in the endoplasmic reticulum, with PDI (7) and Hsp47 (8) as two proteins that have been implicated in the assembly of a procollagen trimer.A fundam...

Recombinant Human Collagen and Biomimetic Variants Using a De Novo Gene Optimized for Modular Assembly

“…72 Yeast systems are a particularly good choice for recombinant human collagen production because they can be engineered to hydroxylate prolines and can yield high expression levels of collagen. Prior studies have shown that P. pastoris 55,73,74 and S. cereVisiae 28,39,75 can produce hydroxylated human collagen types I, II, and III. 39,55,73,76 In fact, commercially available recombinant human collagen III uses a P. pastoris expression system 77 based on cDNA.…”

Applications of Collagen in Medical Devices

“…On the other hand, several groups have reported the successful production of hydroxylated collagen using Saccharomyces systems (33)(34)(35). This can be achieved, for example, by using non-integrating, stable vectors.…”

“…Other arrangements of the 3 genes on these vectors are also effective (36). Alternatively, the 3 genes can be introduced as multiple copies integrated into the yeast genome, all under the control of GAL promoters (35). This approach leads to excellent yields of collagen that has a high degree of prolyl hydroxylation, as shown by a stability (melting temperature) comparable to native, tissue derived collagen (35).…”

Bioengineered collagens

“…Notable progress has been made on commercial, recombinant production of human collagens. 7,[10][11][12][13][14][15][16][17] However, the rapidly emerging technologies that have characterized and produced recombinant non-animal (bacterial) collagens in E. coli [23][24][25]27,28 have introduced an exciting new collagen option that can be readily customized for specific biomedical applications. 28,33 These customized collagens will pave the way for more diverse applications in medical devices, implantable materials and potentially as scaffolds for stem cells, to name but a few examples of uses for these recombinant bacterial collagens.…”

“…7 On the other hand, yeast systems were considerably more promising, and several have been used successfully for the expression of full-length hydroxylated human collagens ( Table 1). [10][11][12] The advantage of the yeasts is they can be optimized to integrate and co-express 3 or 4 genes concurrently. The most successful application, which has provided commercial opportunities, has emerged from expression in P. pastoris ( Table 1).…”