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William C. Raschke

Bio: William C. Raschke is an academic researcher from Salk Institute for Biological Studies. The author has contributed to research in topics: Cell culture & Pichia pastoris. The author has an hindex of 14, co-authored 18 publications receiving 2384 citations.

Papers
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Journal ArticleDOI
TL;DR: The Pichia pastoris heterologous gene expression system has been utilized to produce attractive levels of a variety of intracellular and extracellular proteins of interest and improvements in understanding and application have improved its utility even further.
Abstract: The Pichia pastoris heterologous gene expression system has been utilized to produce attractive levels of a variety of intracellular and extracellular proteins of interest. Recent advances in our understanding and application of the system have improved its utility even further. These advances include: (1) methods for the construction of P. pastoris strains with multiple copies of AOX1-promoter-driven expression cassettes; (2) mixed-feed culture strategies for high foreign protein volumetric productivity rates; (3) methods to reduce proteolysis of some products in high cell-density culture media; (4) tested procedures for purification of secreted products; and (5) detailed information on the structures of N-linked oligosaccharides on P. pastoris secreted proteins. In this review, these advances along with basic features of the P. pastoris system are described and discussed.

993 citations

Journal ArticleDOI
01 Sep 1978-Cell
TL;DR: Secretion of infectious Abelson leukemia virus by two of the cloned cell lines provides conclusive evidence that the Abelson virus is capable of productively infecting the macrophage cell type.

866 citations

Journal ArticleDOI
01 Sep 1980-Cell
TL;DR: This study compares the structures of the secreted and membrane-bound mu (micron) heavy chains by peptide mapping, micro-sequence and carboxypeptidase analyses, and suggests that the micron and microsecond chains from a given B cell are identical except for their 41 and 20 residue C-terminal segments, respectively.

131 citations

Journal ArticleDOI
TL;DR: The use of the methylotrophic industrial yeast Pichia pastoris as a host system for the large scale production of the KPI domain of PN-2AβPP, a secreted isoform of the Alzheimer's amyloid β-protein precursor that contains the Kunitz-type protease inhibitor (KPI) domain, is described.

74 citations

Journal ArticleDOI
TL;DR: The pattern of inhibition among different lymphosarcoma lines suggests that they represent tumors of B lymphocytes at different stages of differentiation, similar to other hematopoietic cell lines.

66 citations


Cited by
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Journal ArticleDOI
16 Feb 1990-Science
TL;DR: It is demonstrated that P210bcr/abl expression can induce chronic myelogenous leukemia and retrovirus-mediated expression of the protein provides a murine model system for further analysis of the disease.
Abstract: In tumor cells from virtually all patients with chronic myelogenous leukemia, the Philadelphia chromosome, a fusion of chromosomes 9 and 22, directs the synthesis of the P210bcr/abl protein. The protein-tyrosine kinase activity and hybrid structure of P210bcr/abl are similar to the oncogene product of the Abelson murine leukemia virus, P160gag/v-abl, which induces acute lymphomas. To determine whether P210bcr/abl can induce chronic myelogenous leukemia, murine bone marrow was infected with a retrovirus encoding P210bcr/abl and transplanted into irradiated syngeneic recipients. Transplant recipients developed several hematologic malignancies; prominent among them was a myeloproliferative syndrome closely resembling the chronic phase of human chronic myelogenous leukemia. Tumor tissue from diseased mice harbored the provirus encoding P210bcr/abl. These results demonstrate that P210bcr/abl expression can induce chronic myelogenous leukemia. Retrovirus-mediated expression of the protein provides a murine model system for further analysis of the disease.

2,199 citations

Journal ArticleDOI
TL;DR: This paper reviews the P. pastoris expression system: how it was developed, how it works, and what proteins have been produced and describes new promoters and auxotrophic marker/host strain combinations which extend the usefulness of the system.
Abstract: During the past 15 years, the methylotrophic yeast Pichia pastoris has developed into a highly successful system for the production of a variety of heterologous proteins. The increasing popularity of this particular expression system can be attributed to several factors, most importantly: (1) the simplicity of techniques needed for the molecular genetic manipulation of P. pastoris and their similarity to those of Saccharomyces cerevisiae, one of the most well-characterized experimental systems in modern biology; (2) the ability of P. pastoris to produce foreign proteins at high levels, either intracellularly or extracellularly; (3) the capability of performing many eukaryotic post-translational modifications, such as glycosylation, disulfide bond formation and proteolytic processing; and (4) the availability of the expression system as a commercially available kit. In this paper, we review the P. pastoris expression system: how it was developed, how it works, and what proteins have been produced. We also describe new promoters and auxotrophic marker/host strain combinations which extend the usefulness of the system.

2,048 citations

Journal ArticleDOI
01 Jul 1983-Nature
TL;DR: A monoclonal antibody specific for a lymphocyte surface molecule that appears to mediate recognition of lymph node HEV and to be required for lymphocyte homing into lymph nodes in vivo is described.
Abstract: Lymphocytes migrate from the bloodstream by recognizing and binding to specialized endothelial cells lining the high endothelial venules (HEV) in lymph nodes and Peyer's patches. We describe here a monoclonal antibody, MEL-14, specific for a lymphocyte surface molecule that appears to mediate recognition of lymph node HEV, and to be required for lymphocyte homing into lymph nodes in vivo.

1,525 citations

Journal ArticleDOI
01 Mar 2005-Yeast
TL;DR: The Pichia pastoris expression system is being used successfully for the production of various recombinant heterologous proteins and the importance of optimizing the physicochemical environment for efficient and maximal recombinant protein production in bioreactors and the role of process control in optimizing protein production is reviewed.
Abstract: The Pichia pastoris expression system is being used successfully for the production of various recombinant heterologous proteins. Recent developments with respect to the Pichia expression system have had an impact on not only the expression levels that can be achieved, but also the bioactivity of various heterologous proteins. We review here some of these recent developments, as well as strategies for reducing proteolytic degradation of the expressed recombinant protein at cultivation, cellular and protein levels. The problems associated with post-translational modifications performed on recombinant proteins by P. pastoris are discussed, including the effects on bioactivity and function of these proteins, and some engineering strategies for minimizing unwanted glycosylations. We pay particular attention to the importance of optimizing the physicochemical environment for efficient and maximal recombinant protein production in bioreactors and the role of process control in optimizing protein production is reviewed. Finally, future aspects of the use of the P. pastoris expression system are discussed with regard to the production of complex membrane proteins, such as G protein-coupled receptors, and the industrial and clinical importance of these proteins.

1,237 citations

Journal ArticleDOI
15 Aug 1986-Science
TL;DR: The 2.8 A resolution three-dimensional structure of a complex between an antigen (lysozyme) and the Fab fragment from a monoclonal antibody against lysozyme has been determined and refined by x-ray crystallographic techniques.
Abstract: The 2.8 A resolution three-dimensional structure of a complex between an antigen (lysozyme) and the Fab fragment from a monoclonal antibody against lysozyme has been determined and refined by x-ray crystallographic techniques. No conformational changes can be observed in the tertiary structure of lysozyme compared with that determined in native crystalline forms. The quaternary structure of Fab is that of an extended conformation. The antibody combining site is a rather flat surface with protuberances and depressions formed by its amino acid side chains. The antigen-antibody interface is tightly packed, with 16 lysozyme and 17 antibody residues making close contacts. The antigen contacting residues belong to two stretches of the lysozyme polypeptide chain: residues 18 to 27 and 116 to 129. All the complementarity-determining regions and two residues outside hypervariable positions of the antibody make contact with the antigen. Most of these contacts (10 residues out of 17) are made by the heavy chain, and in particular by its third complementarity-determining region. Antigen variability and antibody specificity and affinity are discussed on the basis of the determined structure.

1,208 citations