Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Using tissue cell culture to make a protein or polypeptide
Patent
1998-04-06
2000-02-01
Smith, Lynette R. F.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Using tissue cell culture to make a protein or polypeptide
435419, 435468, 435 691, 435 698, 536 235, 536 2353, 536 241, C12N 1500, C12N 1529, C12N 1582
Patent
active
06020169&
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
This invention is related to the field of methods and compositions for recombinant production of foreign polypeptides, and more particularly to production of secreted foreign polypeptides in plant cell culture.
BACKGROUND OF THE INVENTION
Transgenic plant cells have been studied over the past several years for potential use in low cost production of high quality, biologically active mammalian proteins (Sijmons et al., Bio/Technology 8:217-221, 1990; Vandekerckhove et al., Bio/Technology 7:929-932, 1989; Conrad and Fiedler, Plant Molecular Biology 26:1023-1030, 1994; and Ma et al., Science 268:716-719, 1995). Of the various mammalian proteins studied to date, monoclonal antibodies (MAb) have received the most attention because of their potential value as therapeutic and clinical reagents (reviewed in Conrad and Fiedler, Plant Molecular Biology 26:1023-1030, 1994). Initially, individual heavy and light chains were expressed in transgenic tobacco plants, but the expression obtained was exceedingly low (Hiatt et al., Nature 342:76-78, 1989). Although it was well known that large macromolecules such as protein multimers do not readily pass through plant cell membranes (Milburn, Water Flow in Plants, Longman, London, 1979; Carpita et al., Science 205:1144-1147, 1979), two transgenic tobacco plants each expressing either the heavy or light immunoglobulin chains were crossed generating a hybrid plant that coexpressed both chains. The coexpression of both chains led to a large accumulation of antibody within the leaf tissue, an increase from 0.3% to 1.3% of the total leaf protein. Importantly, the antibody retained all its functional and binding capacities. More recently, through successive sexual crosses, it has been possible to generate a functional dimer IgA antibody containing both the J chain and secretory unit (Ma et al., Science 268:716-719, 1995). The antibody accumulated at high levels in the protoplastic or apoplastic space in leaf tissue, with antibody accumulation apparently dependent both upon the stabilizing effect of the heavy/light chain dimer formation and on the presence of functional leader sequences, which appeared to be necessary for assembly. Intact functional antibodies can be produced intracellularly in transgenic plants, but intracellular accumulation requires expensive purification of the antibodies from other cellular proteins.
Other studies have demonstrated that functional IgM antibodies could be generated simply by incorporating both heavy and light chain cDNAs into a single construct rather than crossing individual plants possessing the transgene for either the light or heavy chain (During et al., Plant Mol. Biol. 15:281-293, 1990). Again, accumulation of the multimeric antibody occurred only intracellularly within the leaf tissue. Nevertheless, the studies did underscore the finding that a leader sequence was necessary for accumulation of the antibody. The source of the leader sequence (mammalian or plant) appeared to play a major role in determining the location of protein accumulation within the cell.
One of the first foreign proteins to be expressed and secreted into the medium from a plant cell was human serum albumin (Sijmons et al., Bio/Technology 8:217-221, 1990). The albumin was detected in the culture medium at a level of 0.25 .mu.g/mg of plant protein and appeared to represent the majority of the synthesized albumin suggesting almost all of it was secreted. Unfortunately, because albumin lacks a measurable enzymatic or biological activity, it was not possible to assess the functional capacity of the albumin produced in the plant.
Secretion of functional antibody through the plasma membrane of plant cells has been reported for protoplasts isolated from transgenic plants and for callus cells adapted to suspension culture (Hein et al., Biotechnol. Prog. 7:455-561, 1991). However, the levels of secreted antibody detected in both culture systems were extremely low. In other studies, cultured tobacco cells were transformed with a gene encoding a synthetic anti
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An Gynheung
Lee James M.
Magnuson Nancy S.
Reeves Raymond
Haas Thomas
Smith Lynette R. F.
Washington State University Research Foundation
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