Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Patent
1995-03-14
1997-09-09
Knode, Marian C.
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
5303871, 5303873, 435 696, 435 711, 4351738, 4352523, C12P 2108, C07K 1600, C12N 1513, C12N 1569
Patent
active
056658664
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention provides an improvement to a process for obtaining antibodies in soluble, correctly folded and assembled form.
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 and the time and cost involved in generating the amounts of 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 approval. 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.
A further problem is that proteins produced in bacterial cells are often precipitated as insoluble aggregates within the bacterial cell. This problem has been addressed in a number of ways in the prior art. For example, a large number of patent specifications teach solubilisation of aggregates by the use of chaotropic denaturants and subsequent renaturation. These procedures involve the use of expensive denaturing chemicals, are time-consuming and introduce chemical agents into the production process of which clearance demonstration will be required by the regulatory authorities if the product is destined for clinical use.
An alternative approach has involved attempting to secrete the heterologous protein from the bacteria into the culture medium.
A number of recent advances have been made in bacterial protein expression, both relating to secretion and non-secretion systems. It has been observed, by a number of groups working in this field, that expression
REFERENCES:
patent: 5015573 (1991-05-01), Yarranton et al.
Cabilly, "Growth at sub-optimal temperatures allows the production of functional, antigen-binding Fab fragments in Escherichia coli", Gene, vol. 85, pp. 553-557, 1989.
Schein et al., "Formation of soluble recombinant proteins in Escherichia coli is favored by lower growth temperature", Bio/Technology, vol. 6, pp. 291-294, 1988.
Wright et al., "Dual-origin plasmids containing an amplifiable ColE1 ori; temperature-controlled expression of cloned genes", Gene, vol. 49, pp. 311-321, 1986.
Roberts et al., "Generation of an antibody with enhanced affinity and specificity for its antigen by protein engineering", Nature, vol. 328, pp. 731-734, 1987.
Chalmers et al., "Effects of Temperature on Escheichia coli Overproducing .beta.-Lactamase or Human Epidermal Growth Factor", Appl. Environ. Microbiol., vol. 56, pp. 104-111, 1990.
Bailey Neil Andrew
Weir Andrew Neil Charles
Celltech Therapeutics Limited
Johnson Nancy A.
Knode Marian C.
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