Engineering and construction of expression systems for the production of recombinant pharmaceutical proteins.

摘要

Although the ability to express recombinant proteins for academic and industrial research is a well-established practice, the production of proteins for clinical and manufacturing purposes can be a considerably more complex process. Many times in the case of the latter, numerous additional concerns must be addressed. The work presented in this thesis describes the construction of expression systems to produce two recombinant proteins for use in pharmaceutical manufacturing processes. In one case, recombinant human hemoglobin, the protein was being manufactured for therapeutic use as a blood substitute. In the case of recombinant bovine thrombin, the protein is a key raw material used in the manufacture of recombinant Protein C or Xigris(TM), the only approved pharmaceutical treatment for severe sepsis. As the manufacturing processes were being developed, it was evident that these processes had to be changed. These changes were in the form of engineering modifications to the expression systems. These two proteins also provided distinctly unique opportunities for engineering.;For recombinant human hemoglobin, the expression host was the primary target for engineering. More specifically, Escherichia coli strains were constructed that addressed several issues. These included the accumulation of non-functional product, the availability of a key cofactor, and the ability of the expression host to resist phage contamination. In addition, other potentially beneficial modifications were examined including transcriptional and translational influences. This work resulted in an expression host that was much more amenable to the manufacture of the therapeutic protein.;In the case of recombinant bovine thrombin, the protein itself was the focus of the engineering work. Several engineered forms of the molecule were constructed that demonstrated the protein could be expressed in E. coli as granules, isolated, refolded and subsequently self activated. This engineering and expression work was then extended by the construction of mammalian cells lines that produced the recombinant thrombin precursors. Cell lines expressing the prethrombin-2 precursor generated active thrombin upon secretion. A purification scheme was developed for the efficient isolation of the expressed material. Subsequent characterization of the recombinant enzyme demonstrated that it was equivalent to the animal derived enzyme currently used in the Protein C manufacturing process.