PubMed · 13334632
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F MARKWARDT. 1956. [Studies on hirudin].. https://pubmed.ncbi.nlm.nih.gov/13334632/
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The conformation of a new recombinant RGD-hirudin, which has the activities of anti-thrombin and anti-platelet aggregation, was investigated by multi-dimensional NMR spectroscopy. The 1H NMR spectra of this protein are assigned in a sequential manner by using a combination of 2D NMR techniques to demonstrate through-bond and through-space (<5 A) connectivities. The secondary structure of recombinant RGD-hirudin was deduced from chemical shift indices, sequential NOEs and 3J(HNalpha) coupling constants. The results show that the recombinant RGD-hirudin has two anti-parallel beta-sheets and no alpha-helix, and also that the Arg-Gly-Asp (RGD) binding motif of this protein is located at the end of a long arm, which consists of two anti-parallel beta-strands (residues 26-31 and 36-41). As the strands are connected by a beta-turn, the recombinant RGD-hirudin acquires high flexibility and inhibits platelet aggregation more effectively.
BACKGROUND: After introducing the specific thrombin inhibitor recombinant hirudin (r-hirudin) into clinical practice in cases of heparin-induced thrombocytopenia (HIT, type II) the possibility of its use as an anticoagulant during haemodialysis treatment in HIT II patients is being discussed more frequently. On the one hand, the efficient, safe and routine use of r-hirudin during haemodialyses, including the maintenance of a therapeutic blood level, presupposes that no r-hirudin will leave the circulation by passing through the dialyser membrane. On the other hand, it is important to have dialysers whose permeability to r-hirudin allows its efficient removal from the human body because, to date, no antidote is commercially available in cases of dangerously high blood concentrations of r-hirudin. METHODS: An in vitro circulation model was used to study the r-hirudin permeability of some low- and high-flux dialysers. As r-hirudin-containing vehicles, both albumin-containing saline solution and bovine blood were circulated in the blood space of the system for 2 h. Transmembrane r-hirudin passage was tested by measuring r-hirudin concentration both in the blood and dialysate space fluids using the ecarin clotting time (ECT). RESULTS: Low-flux dialysers with membranes made from polysulfone or regenerated cellulose proved to be almost impermeable to r-hirudin. In contrast, other low-flux membranes were partly permeable to r-hirudin (e.g. Hemophan) or even almost completely permeable (e.g. cellulose acetate). All high-flux dialysers tested were permeable to r-hirudin. CONCLUSIONS: Only low-flux dialysers with polysulfone or regenerated cellulose membranes proved to be suitable for r-hirudin use in routine haemodialysis therapy. Other low-flux, and all high-flux, capillaries are permeable to r-hirudin and offer the possibility of lowering toxic r-hirudin concentrations after overdosing.
This review concerns the issues involved in the industrial development of fed-batch culture processes with Saccharomyces cerevisiae strains producing heterologous proteins. Most of process development considerations with fed-batch recombinant cultures are linked to the reliability and reproducibility of the process for manufacturing environments where quality assurance and quality control aspects are paramount. In this respect, the quality, safety and efficacy of complex biologically active molecules produced by recombinant techniques are strongly influenced by the genetic background of the host strain, genetic stability of the transformed strain and production process factors. An overview of the recent literature of these culture-related factors is coupled with our experience in yeast fed-batch process development for producing various therapeutic grade proteins. The discussion is based around three principal topics: genetics, microbial physiology and fed-batch process design. It includes the fundamental aspects of yeast strain physiology, the nature of the recombinant product, quality control aspects of the biological product, features of yeast expression vectors, expression and localization of recombinant products in transformed cells and fed-batch process considerations for the industrial production of Saccharomyces cerevisiae recombinant proteins. It is our purpose that this review will provide a comprehensive understanding of the fed-batch recombinant production processes and challenges commonly encountered during process development.