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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...

had greater heparin binding capacities in vitro and were cleared more rapidly from the plasma of whole animals. Taken together, these data better define how closely related proteins such as BPI and LBP can have opposing effects on the body's response to LPS. Bacterial endotoxin or lipopolysaccharide (LPS),1 a major component of the outer membrane of Gram-negative bacteria, is a potent mediator of the inflammatory response. Because Gram-negative sepsis remains one of the primary causes of severe systemic inflammation in hospitalized and immunocompromised patients, there is great interest in characterizing proteins involved in the biological response to LPS. In this paper, we focus on two LPS-binding proteins, lipopolysaccharide-binding protein (LBP) and bactericidal/permeability-increasing protein (BPI). LBP and BPI are members of a family of lipid transfer/lipopolysaccharide-binding proteins that also includes cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP). These proteins share significant sequence homology and all bind lipophilic substrates (1).Involved in a complex array of responses to LPS, LBP is a 60-kDA serum glycoprotein that binds the lipid A portion of the LPS molecule to form a high affinity LBP⅐LPS complex (2). This complex potentiates the cellular response to LPS via interaction with the monocytic differentiation antigen CD14 (3, 4). LPS can be transferred from LBP to CD14 (3, 4), present as either a membrane-bound protein on myeloid cells or a soluble serum protein that interacts with endothelial and some epithelial cell lines to elicit an inflammatory response. Recent evidence suggests that LBP may additionally be involved in the neutralization of LPS via interaction with serum lipoproteins (5, 6) or through the internalization of a LBP⅐LPS⅐CD14 complex by neutrophils (7).BPI is a 55-kDa protein found in granules of mature neutrophils and, like LBP, interacts with LPS to form a high affinity complex. BPI, however, binds LPS with higher affinity than does LBP (8 -10), and BPI⅐LPS complexes do not stimulate monocytes or endothelial cells (11-13). The binding of LPS by BPI also prevents binding to LBP, neutralizing the inflammatory activity of LPS (14 -16). BPI and its recombinant N-terminal fragments have been demonstrated to provide protection against challenge with bacteria or purified bacterial endotoxin in several animal models (17-22) and, more recently, in human clinical trials (23). Additionally, Rogy et al. (22) have tested a protein chimera of BPI and LBP, NCY103, in an endotoxin challenge model in baboons.Both BPI and LBP contain 456 amino acids and show an approximate 45% homology at the amino acid level which is distributed over the entire protein sequence. Interestingly, the genes for BPI and LBP lie adjacent to each other in the human genome, suggesting that they might have arisen from a gene duplication event (24). LPS binding is a property of the Nterminal half of both LBP and BPI (9,16,[25][26][27], and a proteolytic N-terminal fragment of BPI (26...

Substantial evidence supports the role of the procollagen C-propeptide in the initial association of procollagen polypeptides and for triple helix formation. To evaluate the role of the propeptide domains on triple helix formation, human recombinant type I procollagen, pN-collagen (procollagen without the C-propeptides), pC-collagen (procollagen without the N-propeptides), and collagen (minus both propeptide domains) heterotrimers were expressed in Saccharomyces cerevisiae. Deletion of the N-or C-propeptide, or both propeptide domains, from both pro␣-chains resulted in correctly aligned triple helical type I collagen. Protease digestion assays demonstrated folding of the triple helix in the absence of the N-and C-propeptides from both pro␣-chains. This result suggests that sequences required for folding of the triple helix are located in the helical/ telopeptide domains of the collagen molecule. Using a strain that does not contain prolyl hydroxylase, the same folding mechanism was shown to be operative in the absence of prolyl hydroxylase. Normal collagen fibrils were generated showing the characteristic banding pattern using this recombinant collagen. This system offers new opportunities for the study of collagen expression and maturation.Collagen is the single most abundant protein found in animals. In the human body, it is expressed in most tissues and plays a structural, as well as a signaling, role in the development, maintenance, and repair of tissues and organs. 20 different collagen types are coded by more than 30 genes. Assembly of trimeric collagen intracellularly and formation of collagen fibers in the extracellular matrix is the result of a complex multistep process (1, 2). Within the endoplasmic reticulum, the individual procollagen polypeptides undergo several co-and post-translational modifications, including hydroxylation of specific prolyl and lysyl residues, selection and alignment of three procollagen polypeptides, and disulfide bond formation among the C-propeptides. Experimental evidence suggests folding of the triple helix begins at the C terminus and propagates toward the N terminus. Prior to triple helix formation, prolyl hydroxylase converts proline in the Y position of GXY triplets to hydroxyproline. Hydrogen bonding between the ␣-chains of the triple helix increases the denaturation temperature of the molecule, preventing it from unfolding at the animal's body temperature (3). Triple helical procollagen is secreted from the cell, and the N-and C-terminal propeptides are removed by specific N-and C-proteinases. The resulting collagen monomers, consisting of triple helical and telopeptide regions, undergo a self-assembly process to generate collagen fibril intermediates and then mature collagen fibers. These fibers are further stabilized by covalent cross-links within the triple helix and telopeptide regions, providing strength and support to the surrounding tissue.

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