Ronald A. Hitzeman

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

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 heat-stable heterotrimers inSaccharomyces. 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.

Production of Human Type I Collagen in Yeast Reveals Unexpected New Insights into the Molecular Assembly of Collagen Trimers

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 proα-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 proα-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.

Automatic Eukaryotic Artificial Chromosomes: Possible Creation of Bacterial Organelles in Yeast

Publisher Summary This chapter discusses the automatic eukaryotic artificial chromosomes (AEAC). Evolutionary theory proposes that mitochondria, plastids, and chloroplasts originated by the engulfment or cell fusion of prokaryotes by eukaryotes. As this endo-symbiotic relationship evolved, the size of the bacterial DNA genome decreased and the functions of genes lost from the bacterial genome were assumed by the eukaryotic chromosome. These experiments demonstrate a new technology that could be used for transferring an entire prokaryotic genome or other large DNA molecules, in circular form, into a eukaryotic cell, where the circular DNA is automatically converted into an artificial linear chromosome in vivo. The production of AEAC-bacterial genomes for animals, human cells (AHACs), and plants preferably require different telomeres, centromeres, and selectable markers that function in these cells to make automatic chromosomes. AHACs carrying genomic or cDNAs for the necessary genes to be transferred would be ideal because of their formation of automatic functional chromosomes upon reaching the nuclei of the human cells.

Automatic Eukaryotic Artificial Chromosomes

Publisher Summary This chapter discusses the automatic eukaryotic artificial chromosomes (AEAC). Evolutionary theory proposes that mitochondria, plastids, and chloroplasts originated by the engulfment or cell fusion of prokaryotes by eukaryotes. As this endo-symbiotic relationship evolved, the size of the bacterial DNA genome decreased and the functions of genes lost from the bacterial genome were assumed by the eukaryotic chromosome. These experiments demonstrate a new technology that could be used for transferring an entire prokaryotic genome or other large DNA molecules, in circular form, into a eukaryotic cell, where the circular DNA is automatically converted into an artificial linear chromosome in vivo. The production of AEAC-bacterial genomes for animals, human cells (AHACs), and plants preferably require different telomeres, centromeres, and selectable markers that function in these cells to make automatic chromosomes. AHACs carrying genomic or cDNAs for the necessary genes to be transferred would be ideal because of their formation of automatic functional chromosomes upon reaching the nuclei of the human cells.

Chapter 22 – Automatic Eukaryotic Artificial Chromosomes: Possible Creation of Bacterial Organelles in Yeast

Publisher Summary This chapter discusses the automatic eukaryotic artificial chromosomes (AEAC). Evolutionary theory proposes that mitochondria, plastids, and chloroplasts originated by the engulfment or cell fusion of prokaryotes by eukaryotes. As this endo-symbiotic relationship evolved, the size of the bacterial DNA genome decreased and the functions of genes lost from the bacterial genome were assumed by the eukaryotic chromosome. These experiments demonstrate a new technology that could be used for transferring an entire prokaryotic genome or other large DNA molecules, in circular form, into a eukaryotic cell, where the circular DNA is automatically converted into an artificial linear chromosome in vivo. The production of AEAC-bacterial genomes for animals, human cells (AHACs), and plants preferably require different telomeres, centromeres, and selectable markers that function in these cells to make automatic chromosomes. AHACs carrying genomic or cDNAs for the necessary genes to be transferred would be ideal because of their formation of automatic functional chromosomes upon reaching the nuclei of the human cells.