The molecular basis of blood coagulation

The use of the methylotrophic yeast, Pichia pastoris, as a cellular host for the expression of recombinant proteins has become increasing popular in recent times. P. pastoris is easier to genetically manipulate and culture than mammalian cells and can be grown to high cell densities. Equally important, P. pastoris is also a eukaryote, and thereby provides the potential for producing soluble, correctly folded recombinant proteins that have undergone all the post-translational modifications required for functionality. Additionally, linearized foreign DNA can be inserted in high efficiency via homologous recombination procedures to generate stable cell lines whilst expression vectors can be readily prepared that allow multiple copies of the target protein, multimeric proteins with different subunit structures, or alternatively the target protein and its cognate binding partners, to be expressed. A further benefit of the P. pastoris system is that strong promoters are available to drive the expression of a foreign gene(s) of interest, thus enabling production of large amounts of the target protein(s) with relative technical ease and at a lower cost than most other eukaryotic systems. The purpose of this review is to summarize important developments and features of this expression system and, in particular, to examine from an experimental perspective the genetic engineering, protein chemical and molecular design considerations that have to be taken into account for the successful expression of the target recombinant protein. Included in these considerations are the influences of P. pastoris strain selection; the choice of expression vectors and promoters; procedures for the transformation and integration of the vectors into the P. pastoris genome; the consequences of rare codon usage and truncated transcripts; and techniques employed to achieve multi-copy integration numbers. The impact of the alcohol oxidase (AOX) pathways in terms of the mut+ and muts phenotypes, intracellular expression and folding pathways is examined. The roles of pre–pro signal sequences such as the alpha mating factor (α-MF) and the Glu–Ala repeats at the kex2p cleavage site on the processing of the protein translate(s) have also been considered. Protocols for the generation of protein variants and mutants for screening for orphan cognate binding partners and the use of experimental platforms addressing the molecular recognition behaviour of recombinant proteins such as the extracellular domains of transmembrane receptors with their physiological ligands are also described. Finally, the palindromic patterns of glycosylation that can occur with these expression systems, in terms of the role and location of the sequon in the primary structure, the number of mannose units and the types of oligosaccharides incorporated as Asn- or O-linkages and their impact on the thermostability and immunogenicity of the recombinant protein are considered. Procedures to prevent glycosylation through manipulation of cell culture conditions or via enzymatic and site-directed mutagenesis methods are also discussed. Copyright © 2004 John Wiley & Sons, Ltd.

Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production

The molecular mechanisms that finely co-ordinate fibrin formation and fibrinolysis are now well defined. The structure and function of all major fibrinolytic proteins, which include serine proteases, their inhibitors, activators and receptors, have been characterized. Measurements of real time, dynamic molecular interactions during fibrinolysis of whole blood clots can now be carried out in vitro. The development of gene-targeted mice deficient in one or more fibrinolytic protein(s) has demonstrated expected and unexpected roles for these proteins in both intravascular and extravascular settings. In addition, genetic analysis of human deficiency syndromes has revealed specific mutations that result in human disorders that are reflective of either fibrinolytic deficiency or excess. Elucidation of the fine control of fibrinolysis under different physiological and pathological haemostatic states will undoubtedly lead to novel therapeutic interventions. Here, we review the fundamental features of intravascular plasmin generation, and consider the major clinical syndromes resulting from abnormalities in fibrinolysis.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-2141.2005.05444.x

Molecular mechanisms of fibrinolysis.

Basic and clinical aspects of fibrinolysis and thrombolysis.

The yeast secretory pathway is perturbed by mutations in PMR1, a member of a Ca2+ ATPase family

An Escherichia coli library containing cDNA sequences derived from mRNA of human pharnygeal carcinoma cells was constructed using the plasmid pBR322. The library was screened with a set of 8 deoxyoligonucleotides, 14 bases in length, which collectively code for the amino acid sequence …Met-Tyr-Asn-Asp-Pro… found in human urokinase (u-PA). A plasmid containing an ∼1700 base pair cDNA insert coding for the carboxy terminal sequence of u-PA was identified. The 5′ region was obtained by priming poly(A) mRNA with a DNA fragment derived from the initial clone. An expression vector was constructed which directs the synthesis of pro u-PA in E. coli.

Cloning and Expression of the Gene for Pro-urokinase in Escherichia coli

Coronary thrombolysis by intravenous infusion of recombinant single chain urokinase-type plasminogen activator or recombinant urokinase in baboons: Effect on regional blood flow, infarct size and hemostasis

Wolfgang A. Guenzler

Papers

Cloning and Expression of the Gene for Pro-urokinase in Escherichia coli

Coronary thrombolysis by intravenous infusion of recombinant single chain urokinase-type plasminogen activator or recombinant urokinase in baboons: Effect on regional blood flow, infarct size and hemostasis

Neue als plasminogenaktivatoren einsetzbare polypeptide, dafuer codierende plasmide und verfahren zu deren herstellung und deren verwendung