Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1995-03-14
1999-07-27
Scheiner, Toni R.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
435471, 4352523, 43525233, 435 712, 435 697, 5303873, 5303871, 536 2353, 536 231, 514 12, A61K 39395, C12N 1500
Patent
active
059289031
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention relates to a method for the expression of recombinant antibody genes in bacterial host cells in a defined medium in the absence of antibiotic selection.
DESCRIPTION OF BACKGROUND ART
The rapid developments in recombinant DNA techniques have resulted in the identification and isolation of many novel genes, some of known function and some of unknown function. Invariably there is a need to express the gene in a heterologous cell system in order to produce material for structure-function studies, diagnostic reagents such as monoclonal or polyclonal antibodies and material for in vivo activity testing and therapy.
Several alternative systems for the expression of foreign genes have been developed including systems based upon mammalian cells, insect cells, fungal cells, bacterial cells and transgenic animals or plants. The choice of expression system for a given gene depends upon the likely features of the encoded protein, for example any post-translational protein modifications needed for biological activity, as well as the objective of the study. Other important considerations for the investigator are the facilities available, time and cost involved in generating the amounts or recombinant protein required.
The most widely used and convenient system for the production of foreign proteins remains that based on the prokaryote Escherichia coli. The advantages of this system comprise the ease of gene manipulation, the availability of reagents including gene expression vectors, the ease of producing quantities of protein (up to a gramme in simple shake-flask culture), speed and the high adaptability of the system to express a wide variety of proteins.
Expression of any foreign gene in E. coli begins with the insertion of a cDNA copy of the gene into an expression vector. Many forms of expression vector are available. Such vectors usually comprise a plasmid origin of DNA replication, an antibiotic selectable marker and a promoter and transcriptional terminator separated by a multi-cloning site (expression cassette) and a DNA sequence encoding a ribosome binding site. The method of transcriptional regulation varies between the various promoters now available (ptac, .lambda.pL, T7). The ptac and T7 expression based systems are controlled by the chemical inducer IPTG, whilst the .lambda. promoters are controlled by a temperature switch.
A problem encountered with E. coli based expression systems is the difficulty of producing material which is acceptable for therapeutic use. The use of complex media, antibiotic selection and potentially hazardous inducers such as IPTG may potentially render products such as recombinant antibody fragments produced by E. coli fermentation technology unacceptable to the regulatory authorities for clinical applications. Evidence demonstrating clearance of these agents from the final product must be provided in order to secure regulatory clearance. Clearance of these agents, and especially demonstrating such clearance, is expensive. It is therefore desirable that an expression system should avoid the three above-mentioned problems.
Avoidance of these problems is not straightforward. Plasmids, especially expression vectors, place a metabolic load on the host cell which acts as a selective pressure favouring loss of the plasmid from the cell. Therefore, in order to reduce the possibility of plasmid loss, or rearrangement of the plasmid to delete the expression activity, it is apparent that the metabolic load placed on a cell by other sources should be reduced to a minimum. Therefore, the use of complex media which contain, in addition to essential amino acids and minerals, a variety of naturally-sourced vitamins, cofactors and the like which alleviate the metabolic load on the cell is favoured.
The term "complex medium" is used herein according to its well-known signification in the art, that is to denote a medium the exact formulation and chemical composition of which has not been determined. Frequently, such media are at least partly derived from na
REFERENCES:
patent: 5015573 (1991-05-01), Yarrington et al.
Carter et al., "High Level Escherichia coli Expression and Production of a Bivalent Humanized Antibody Fragment", Bio/Technology, vol. 10, Feb. 1992, pp. 163-167.
Roberts et al., "Generation of an Antibody with Enhanced Affinity and Specificity for its Antigen by Protein Engineering", Nature, vol. 328, Aug. 1987, pp. 731-734.
Hashimoto-Gotoh et al., "Specific-purpose Plasmid Cloning Vectors", Gene, vol. 16, 1981, pp. 227-235.
Wright et al., "Dual-origin Plasmids Containing an Amplifiable ColE1 ori; Temperature-controlled Expression of Cloned Genes", Gene, vol. 49, 1986, pp. 311-321.
Helinski et al., "Partitioning of the pSC101 Plasmid During Cell Division", Plasmids in Bacteria, 1985, pp. 383-395.
Mountain Andrew
Weir Andrew Neil Charles
Celltech Therapeutics Limited
Reeves Julie E
Scheiner Toni R.
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