CROSS REFERENCE TO RELATED APPLICATIONS

This is the U.S. National Stage of International Application No. PCT/GB2017/050028, filed Jan. 9, 2017, which was published in English under PCT Article 21(2), which in turn claims the benefit of GB 1600512.6, filed Jan. 12, 2016.

FIELD OF THE INVENTION

The disclosure relates to recombinant protein expression systems comprising genetically modified cells wherein the cells are transformed or transfected with tRNA genes to reduce base mismatch due to genetic degeneracy in the genetic code and hence reliance on wobble for decoding; the modified cells have so improved recombinant protein expression.

BACKGROUND TO THE INVENTION

The amino acid composition of proteins determines their properties and is encoded by the DNA sequence of genes. The gene sequence is copied into messenger RNA (mRNA) and decoded using transfer RNA (tRNA) that delivers amino acids in the specified order. The code is written in contiguous triplets of bases, called codons and is decoded by binding to the tRNAs' complementary base triplets, called anticodons. Each tRNA has a single anticodon and delivers a specified amino acid. However, although the combination of the four bases present in the DNA provides 64 codons, there are fewer tRNAs and anticodons present.

DNA comprises four different bases whereby adenosine specifically binds with thymine and guanine specifically binds with cytosine. All organisms have fewer species of tRNA than codons and therefore some tRNA species must pair with more than one codon, which led to the “Wobble” hypothesis postulating that the 5′ base on the anticodon, which binds to the 3′ base on the mRNA, can have non-standard base pairing, with only two of the three bases optimally matched. Suboptimal base pairing can lead to misincorporation errors upon translation. The most frequent errors correspond to a G (mRNA)/U (tRNA) base pair mismatch and additional wobble position mismatches (C/U and U/U) during translation.

Heterologous expression of recombinant proteins provided a wealth of diagnostic and therapeutic agents. Often bacterial systems are the preferred host for protein expression due to the low maintenance and easy handling. However, inability to accommodate for posttranslational modifications led to the usage of other expression systems such as yeast, insect cells, plants or mammalian expression systems. Approximately 70% of recombinant protein pharmaceuticals and most proteins used for vaccination, human therapy or diagnostics are currently produced in mammalian cells or cell lines, such as CHO or HEK293. However, although the choice of expression systems aids expression, there are still significant misincorporation levels being reported when expressing therapeutic monoclonal antibodies in CHO cells during clinical development. The impact of such sequence variation affects not just the quality and efficacy, but could compromise patient safety by triggering an immunogenic response.

Optimal protein expression systems leading to error-free and soluble protein expression are highly desired. As described above, no organism harbours a full set of tRNAs and moreover, there are species-dependent differences. Expression of e.g. a human-derived transgene in a bacterial system is often problematic, as the codon usage and frequency can differ greatly, leading to inefficient expression. This problem is generally addressed by optimising codons to the preferred codon usage by the expression hosts. However, manipulating transgene sequence is extremely problematic, as it interferes with the structure, folding, stability and regulation of the encoded mRNA transcript in ways that are poorly understood and highly unpredictable. An alternative approach is to introduce additional tRNA transgenes into the host so as to improve codon recognition; this has been used successfully inE. coli, but has not been attempted in eukaryotic hosts. In addition this approach does not deal with the problems associated with wobble such as inefficient translation and reduced translational fidelity.

The present disclosure relates to the introduction of tRNA genes with anticodons to optimally match codons that otherwise depend on wobble; in this way, unintended heterogeneity can be reduced and efficiency of protein synthesis can be improved.

STATEMENT OF THE INVENTION

According to an aspect of the invention there is provided an isolated eukaryotic cell wherein the cell is modified by transfection or transformation with a nucleic acid molecule comprising a transcription cassette, the transcription cassette comprising at least one nucleotide sequence encoding a transfer RNA [tRNA], the tRNA including an anticodon nucleotide sequence that is absent from said eukaryotic cell, wherein said anticodon sequence corrects base mismatches at any one of the cognate first, second or third nucleotide position in the corresponding messenger RNA [mRNA] codon to improve translation efficiency and/or decrease amino acid mis-incorporation as a consequence of degeneracy in the genetic code.

In an alternative aspect of the invention said cell is a prokaryotic cell.

In a preferred embodiment of the invention said base mismatch correction reduces said cells reliance on wobble during translation of said mRNA.

“Wobble” is a well-known natural phenomenon. The genetic code means that a cell would need tRNAs with 61 different anticodons to complement the available 61 codons. However, due to the degeneracy of the genetic code, the third base is less discriminatory for the amino acid than the other two bases. This third position in the codon is referred to as the “wobble” position. At this position Us and Cs may be read by a G in the anticodon. Similarly, As and Gs may be read by a U or y (pseudouridine) in the anticodon, and A, U or C can be read by inosine in the anticodon. This flexibility in codon recognition can result in less efficient translation and potential mis-incorporation of amino acids. The invention addresses these problems by genetically engineering cells to reduce the reliance on “wobble” to enhance translation and increase translation fidelity. The transcription cassette according to the invention is adapted for expression of said tRNA. This includes the provision of a suitable RNA polymerase III promoter. Alternatively, the transcription cassette can be intergrated into the genome of said cell at a position near to a suitable promoter, for example an RNA polymerase III promoter.

In a preferred embodiment of the invention said tRNA corrects a base mismatch in the first, second or third nucleotide position in the corresponding mRNA codon.

In a preferred embodiment of the invention said nucleic acid molecule is part of an expression vector adapted for eukaryotic expression of said tRNA.

In a preferred embodiment of the invention said cell is further modified by transformation or transfection with a nucleic acid molecule encoding a recombinant protein, polypeptide or peptide.

In a preferred embodiment of the invention said expression vector is adapted for expression in a mammalian cell.

Typically, said adaptation includes the provision of transcription control sequences (promoter sequences) which mediate cell/tissue specific expression. These promoter sequences may be cell/tissue specific, inducible or constitutive. “Promoter” is an art recognised term and, for the sake of clarity, includes the following features which are provided by example only. Enhancer elements are cis acting nucleotide sequences often found 5′ to the transcription initiation site of a gene (enhancers can also be found 3′ to a gene sequence or even located in intronic sequences). Enhancers function to increase the rate of transcription of the gene to which the enhancer is linked. Enhancer activity is responsive to trans acting transcription factors (polypeptides) which have been shown to bind specifically to enhancer elements. The binding/activity of transcription factors is responsive to a number of physiological/environmental cues which include, by example and not by way of limitation, intermediary metabolites (e.g. glucose, lipids), environmental effectors (e.g. heat). Promoter elements also include so called TATA box and RNA polymerase initiation selection sequences which function to select a site of transcription initiation. These sequences also bind polypeptides which function, inter alia, to facilitate transcription initiation selection by RNA polymerase II or III. The vector may be a bi-functional expression vector which functions in multiple hosts. In the case of genomic DNA, this may contain its own promoter or other regulatory elements and in the case of cDNA this may be under the control of an appropriate promoter or other regulatory elements for expression in the host cell.

Adaptations also include the provision of selectable markers and autonomous replication sequences which facilitate the maintenance of said vector in either the eukaryotic cell or prokaryotic host. Vectors which are maintained autonomously are referred to as episomal vectors. Episomal vectors are desirable since these molecules can incorporate large DNA fragments (30-50 kb DNA). Episomal vectors of this type are described in WO98/07876. Alternatively, vectors can be “integrating vectors” which recombine with chromosomal DNA to stably transform the cell into which the integrating vector is incorporated.

Adaptations which facilitate the expression of vector encoded recombinant genes include the provision of transcription termination/polyadenylation sequences. This also includes the provision of internal ribosome entry sites (IRES) which function to maximise expression of vector encoded genes arranged in bi-cistronic or multi-cistronic expression cassettes. Expression control sequences also include so-called Locus Control Regions (LCRs). These are regulatory elements which confer position-independent, copy number-dependent expression to linked genes when assayed as transgenic constructs. LCRs include regulatory elements that insulate transgenes from the silencing effects of adjacent heterochromatin, Grosveld et al., Cell (1987), 51: 975-985. There is a significant amount of published literature with respect to expression vector construction and recombinant DNA techniques in general. Please see, Sambrook et al (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory, Cold Spring Harbour, N.Y. and references therein; Marston, F (1987) DNA Cloning Techniques: A Practical Approach Vol III IRL Press, Oxford UK; DNA Cloning: F M Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

Additionally, a number of viruses are commonly used as vectors for the delivery of exogenous genes. Commonly employed vectors include recombinantly modified enveloped or non-enveloped DNA and RNA viruses, preferably selected from baculoviridiae, parvoviridiae, picornoviridiae, herpesveridiae, poxviridae, adenoviridiae, or picornnaviridiae. Chimeric vectors may also be employed which exploit advantageous elements of each of the parent vector properties (See e.g., Feng, et al. (1997) Nature Biotechnology 15:866-870). Such viral vectors may be wild-type or may be modified by recombinant DNA techniques to be replication deficient, conditionally replicating or replication competent.

In a preferred embodiment of the invention said expression vector is adapted for expression in a fungal cell.

In a preferred embodiment of the invention said expression vector is adapted for expression in an insect cell.

In a preferred embodiment of the invention said expression vector is adapted for expression in a plant cell.

Suitable promoters for expression of recombinant proteins or tRNAs in plant cells include constitutive, tissue-specific, inducible, developmental or other promoters for expression in plant cells. Such promoters include viral, fungal, bacterial, animal and plant-derived promoters capable of functioning in plant cells. Constitutive promoters include, for example CaMV 35S promoter (Odell et al. (1985) Nature 313, 9810-812); rice actin (McElroy et al. (1990) Plant Cell 2: 163-171); ubiquitin (Christian et al. (1989) Plant Mol. Biol. 18 (675-689); pEMU (Last et al. (1991) Theor Appl. Genet. 81: 581-588); MAS (Velten et al. (1984) EMBO J. 3. 2723-2730); ALS promoter (U.S. application Ser. No. 08/409,297), and the like. Other constitutive promoters include those in U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680, 5,268,463; and 5,608,142, each of which is incorporated by reference.

Chemical-regulated plant promoters can be used to modulate the expression of a gene in a plant cell through the application of an exogenous chemical regulator. Depending upon the objective, the promoter may be a chemical-inducible promoter, where application of the chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression. Chemical-inducible promoters are known in the art of plant transgenic expression and include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR-la promoter, which is activated by salicylic acid. Other chemical-regulated plant promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88: 10421-10425 and McNellis et al. (1998) Plant J. 14(2): 247-257) and tetracycline-inducible and tetracycline-repressible promoters (see, for example, Gatz et al. (1991) Mol. Gen. Genet. 227: 229-237, and U.S. Pat. Nos. 5,814,618 and 5,789,156, herein incorporated by reference.

Where enhanced expression in particular tissues is desired, tissue-specific promoters can be utilised. Tissue-specific promoters include those described by Yamamoto et al. (1997) Plant J. 12(2): 255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7): 792-803; Hansen et al. (1997) Mol. Gen. Genet. 254(3): 337-343; Russell et al. (1997) Transgenic Res. 6(2): 157-168; Rinehart et al. (1996) Plant Physiol. 112(3): 1331-1341; Van Camp et al. (1996) Plant Physiol. 112(2): 525-535; Canevascni et al. (1996) Plant Physiol. 112(2): 513-524; Yamamoto et al. (1994) Plant Cell Physiol. 35(5): 773-778; Lam (1994) Results Probl. Cell Differ. 20: 181-196; Orozco et al. (1993) Plant Mol. Biol. 23(6): 1129-1138; Mutsuoka et al. (1993) Proc. Natl. Acad. Sci. USA 90 (20): 9586-9590; and Guevara-Garcia et al (1993) Plant J. 4(3): 495-50.

Particular of interest in the present context are nucleic acid constructs which operate as plant vectors. Specific procedures and vectors previously used with wide success in plants are described by Guerineau and Mullineaux (1993) (Plant transformation and expression vectors. In: Plant Molecular Biology Labfax (Croy R R D ed) Oxford, BIOS Scientific Publishers, pp 121-148. Suitable vectors may include plant viral-derived vectors (see e.g. EP194809). If desired, selectable genetic markers may be included in the construct, such as those that confer selectable phenotypes such as resistance to herbicides (e.g. kanamycin, hygromycin, phosphinotricin, chlorsulfuron, methotrexate, gentamycin, spectinomycin, imidazolinones and glyphosate).

In a preferred embodiment of the invention said isolated eukaryotic cell is an insect, plant, fungal or mammalian cell.

In a preferred embodiment of the invention said isolated mammalian cell is a non-human mammalian cell.

In a further preferred embodiment of the invention said isolated mammalian cell is selected from the group consisting of: Chinese Hamster Ovary, [CHO], HEK293, NS0 or CAP cells.

In a further preferred embodiment of the invention said fungal cell is selected from the group consisting of yeast:Saccharomyces cerevisae, Schizosaccharomyces pombe Pichia pastoris, Aspergillusspp [e.gA. niger],Kluyveromyces lactisandTrichoderma reeseiandYarrowia lipolytica.

In a preferred embodiment of the invention said plant cell is isolated from a plant species selected from the group: corn (Zea mays), canola (Brassica napus, Brassica rapassp.), alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cerale),Sorghum(Sorghum bicolor, Sorghum vulgare), sunflower (helianthus annuas), wheat (Tritium aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Cofeaspp.), coconut (Cocos nucifera), pineapple (Anana comosus), citris tree (Citrusspp.) cocoa (Theobroma cacao), tea (Camellia senensis), banana (Musaspp.), avacado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifer indica), olive (Olea europaea),papaya(Carica papaya), cashew (Anacardium occidentale),macadamia(Macadamia intergrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), oats, barley, vegetables and ornamentals.

Preferably, plant cells of the present invention are crop plants (for example, cereals and pulses, maize, wheat, potatoes, tapioca, rice,Sorghum, millet, cassava, barley, pea, and other root, tuber or seed crops). Important seed crops are oil-seed rape, sugar beet, maize, sunflower, soybean, andSorghum. Horticultural plants to which the present invention may be applied may include lettuce, endive, and vegetable brassicas including cabbage, broccoli, and cauliflower, and carnations and geraniums. The present invention may be applied in tobacco, cucurbits, carrot, strawberry, sunflower, tomato, pepper,chrysanthemum. Grain plants that provide seeds of interest include oil-seed plants and leguminous plants. Seeds of interest include grain seeds, such as corn, wheat, barley, rice,Sorghum, rye, etc. Oil-seed plants include cotton, soybean, safflower, sunflower,Brassica, maize, alfalfa, palm, coconut, etc. Leguminous plants include beans and peas. Beans include guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea.

In a further preferred embodiment of the invention said expression cassette comprises nucleotide sequences encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 different tRNAs.

In an embodiment of the invention said nucleic acid encodes a transfer RNA selected from the group of Ala-tRNA, Arg-tRNA, Asp-tRNA, Asn-tRNA, Cys-tRNA, Glu-tRNA, Gln-tRNA, Gly-tRNA, His-tRNA, Ile-tRNA, Leu-tRNA, Lys-tRNA, Met-tRNA, Phe-tRNA, Pro-tRNA, Ser-tRNA, Thr-tRNA, Trp-tRNA, Tyr-tRNA and Val-tRNA.

In a preferred embodiment said anticodon is cytosine and/or adenine rich.

In a preferred embodiment said anticodon is uracil free.

In a further preferred embodiment said anticodon is selected from the group consisting of AGC, GGC, CGC, UGC, ACC, GCC, CCC, UCC, AGG, GGG, CGG, UGG, AGU, GGU, CGU, UGU, AAC, GAC, CAC, UAC, AAA, GAA, AUU, GUU, CUU, UUU, AUC, GUC, CUC, UUC, AGA, GGA, CGA, UGA, ACU, GCU, ACG, GCG, CCG, UCG, CCU, UCU, AAG, GAG, CAG, UAG, CAA, UAA, AAU, GAU, UAU, CAU, AUA, GUA, ACA, GCA, AUG, GUG, CUG, UUG, CCA

In a preferred embodiment of the invention said modified cell is transformed or transfected with a nucleic acid molecule encoding a therapeutic antibody.

In an alternative embodiment of the invention said modified cell is transformed or transfected with a nucleic acid molecule encoding a pharmaceutical or therapeutic protein or peptide.

In a further preferred embodiment of the invention said modified cell is transformed or transfected with a nucleic acid molecule encoding an industrial enzyme.

In a preferred embodiment of the invention said modified cell is further modified by transformation or transfection with a nucleic acid molecule comprising a 7SL nucleotide sequence.

Secreted and membrane proteins contain signal sequences that are required to target proteins to be secreted to the endoplasmic reticulum. The signal sequences of native and recombinant proteins destined for secretion interact with a signal recognition particle which is a ribonucleoprotein complex comprising six different proteins and a structural RNA of around 300 nucleotides and is referred to as 7SL RNA. 7SL RNA is conserved across species and although there is little homology at the level of sequence identity the secondary structures of 7SL RNA are highly conserved comprising a central double stranded region which is flanked at one end by two small stem loop structures and at the other end by two relatively large stem loop structures. We disclose that co-expression of 7SL nucleic acid in our modified cells greatly enhances expression of recombinant protein.

In a preferred embodiment of the invention said nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO: 10, or a nucleotide sequence variant that has at least 90% nucleotide sequence identity over the full length to the nucleotide sequence set forth in SEQ ID NO: 10.

In a preferred embodiment of the invention there is provided a nucleic acid molecule that comprises a nucleotide sequence that has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity over the full length to the nucleotide sequence set forth in SEQ ID NO: 10.

Preferably, said nucleic acid molecule comprises or consists of a nucleotide sequence as set forth in SEQ ID NO: 10.

In a preferred embodiment of the invention translation efficiency is enhanced by greater than 1-fold when compared to a non-modified cell of the same species.

In a preferred embodiment of the invention translation efficiency is enhanced at least two fold when compared to a non-modified cell of the same species.

Preferably, translational efficiency is enhanced at least 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or at least 10-fold when compared to a non-modified cell of the same species. Preferably, translation is enhanced by greater than 10-fold when compared to a non-modified cell of the same species.

In a preferred embodiment of the invention the mis-incorporation of amino acids in said recombinant protein is suppressed by greater than 1-fold when compared to the amino acid mis-incorporation rate in a non-modified cell of the same species.

In a preferred embodiment of the invention the mis-incorporation of amino acids in said recombinant protein is suppressed by at least 2-fold when compared to the amino acid mis-incorporation rate in a non-modified cell of the same species.

Preferably, mis-incorporation is suppressed by at least 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold or at least 40-fold when compared to the amino acid mis-incorporation rate in a non-modified cell of the same species.

According to an aspect of the invention there is provided a modified cell according to the invention for use in reducing tRNA mismatch as a consequence of the degeneracy in the genetic code in the production of recombinant protein.

Preferably, said reduction in tRNA mismatch reduces said cell's reliance on wobble during translation of said mRNA by said modified cell.

According to a further aspect of the invention there is provided a method for reducing tRNA mismatch as a consequence of the degeneracy in the genetic code comprising the steps:i) providing a modified cell according to the invention;ii) incubating the cell in i) under cell culture conditions suitable for expression of one or more recombinant proteins/peptides expressed by said modified cell; and optionallyiii) isolating the expressed recombinant proteins/peptides from the modified cell or cell culture medium.

According to a further aspect of the invention there is provided a method to customise expression of a selected recombinant protein in a cell expression system comprising the steps:i) analysing the nucleotide sequence of a nucleic acid molecule encoding a recombinant protein to be expressed to identify one or more codon/anticodon tRNA mismatches;ii) transfecting or transforming a cell with one or more transcription cassettes comprising one or more tRNA genes that correct at least one base pairing mismatch in at least one codon to provide a modified cell;iii) transfecting or transforming said modified cell with the recombinant protein to be expressed; and optionallyiv) providing cell culture conditions to express said recombinant protein.

In a preferred method of the invention said cell is a eukaryotic cell.

Preferably, said cell is a mammalian cell, a fungal cell, an insect cell or a plant cell.

In an alternative preferred method of the invention said cell is a prokaryotic cell, for example a bacterial cell.

According to an aspect of the invention there is provided a modified cell obtained or obtainable by the method according to the invention.

According to a further aspect of the invention there is provided a cell culture vessel comprising a modified cell according to the invention. In a preferred embodiment of the invention said cell culture vessel is a bioreactor such as a fermenter.

If eukaryotic microbial or prokaryotic expression is preferred in the methods according to the invention they are grown or cultured in the manner with which the skilled worker is familiar, depending on the host organism. As a rule, microorganisms are grown in a liquid medium comprising a carbon source, usually in the form of sugars, a nitrogen source, usually in the form of organic nitrogen sources such as yeast extract or salts such as ammonium sulfate, trace elements such as salts of iron, manganese and magnesium and, if appropriate, vitamins, at temperatures of between 0° C. and 100° C., preferably between 10° C. and 60° C., while gassing in oxygen.

The pH of the liquid medium can either be kept constant, that is to say regulated during the culturing period, or not. The cultures can be grown batch wise, semi-batch wise or continuously. Nutrients can be provided at the beginning of the fermentation or fed in semi-continuously or continuously. Recombinant protein produced can be isolated from the organisms as described above by processes known to the skilled worker, for example by extraction, if appropriate precipitation with salt, and/or chromatography. To this end, the organisms can advantageously be disrupted beforehand.

In this process, the pH value kept between pH 4 and 12, preferably between pH 6 and 9, especially preferably between pH 7 and 8. The culture medium to be used must suitably meet the requirements of the strains in question. Descriptions of culture media for various microorganisms can be found in the textbook “Manual of Methods for General Bacteriology” of the American Society for Bacteriology (Washington D.C., USA, 1981).

As described above, these media which can be employed in accordance with the invention usually comprise one or more carbon sources, nitrogen sources, inorganic salts, vitamins and/or trace elements. Preferred carbon sources are sugars, such as mono-, di- or polysaccharides. Examples of carbon sources are glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose. Sugars can also be added to the media via complex compounds such as molasses or other by-products from sugar refining. The addition of mixtures of a variety of carbon sources may also be advantageous. Other possible carbon sources are oils and fats such as, for example, soya oil, sunflower oil, peanut oil and/or coconut fat, fatty acids such as, for example, palmitic acid, stearic acid and/or linoleic acid, alcohols and/or polyalcohols such as, for example, glycerol, methanol and/or ethanol, and/or organic acids such as, for example, acetic acid and/or lactic acid.

Nitrogen sources are usually organic or inorganic nitrogen compounds or materials comprising these compounds. Examples of nitrogen sources comprise ammonia in liquid or gaseous form or ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids or complex nitrogen sources such as cornsteep liquor, soya meal, soya protein, yeast extract, meat extract and others. The nitrogen sources can be used individually or as a mixture.

Inorganic salt compounds which may be present in the media comprise the chloride, phosphorus and sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron. Inorganic sulfur-containing compounds such as, for example, sulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfides, or else organic sulfur compounds such as mercaptans and thiols may be used as sources of sulfur for the production of sulfur-containing fine chemicals, in particular of methionine. Phosphoric acid, potassium dihydrogenphosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts may be used as sources of phosphorus.

The fermentation media used according to the invention for culturing microorganisms usually also comprise other growth factors such as vitamins or growth promoters, which include, for example, biotin, riboflavin, thiamine, folic acid, nicotinic acid, panthothenate and pyridoxine. Growth factors and salts are frequently derived from complex media components such as yeast extract, molasses, cornsteep liquor and the like. It is moreover possible to add suitable precursors to the culture medium.

According to a further aspect of the invention there is provided a method for the customised in vitro synthesis of recombinant protein comprising the steps:i) analysing the nucleotide sequence of nucleic acid molecule encoding a recombinant protein to be expressed to identify one or more codon/anticodon tRNA mismatches;ii) providing a reaction mixture comprising mRNA encoding said recombinant protein and one or more tRNAs that correct at least one base pairing mismatch in at least one codon in said mRNA; andiii) providing reaction conditions for the synthesis of said recombinant protein from said mRNA.

In a preferred method of the invention said reaction mixture includes an expression vector and RNA polymerase and the method is a combined transcription/translation reaction.

According to a further aspect of the invention there is provided a kit comprising:an expression vector[s] comprising one or more tRNA genes; optionallyan expression vector adapted to receive one or more recombinant nucleic acid molecules; andenzymes and buffers required for sub-cloning and/or transfection/transformation.

Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of the words, for example “comprising” and “comprises”, means “including but not limited to”, and is not intended to (and does not) exclude other moieties, additives, components, integers or steps. “Consisting essentially” means having the essential integers but including integers which do not materially affect the function of the essential integers.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

PREFERRED EMBODIMENTS

Antibodies include polyclonal, monoclonal antibodies, humanised and chimeric antibodies and derived fragments comprising CDRs.

Chimeric antibodies are recombinant antibodies in which all of the V-regions of a mouse or rat antibody are combined with human antibody C-regions. Humanised antibodies are recombinant hybrid antibodies which fuse the complementarity determining regions from a rodent antibody V-region with the framework regions from the human antibody V-regions. The C-regions from the human antibody are also used. The complementarity determining regions (CDRs) are the regions within the N-terminal domain of both the heavy and light chain of the antibody to where the majority of the variation of the V-region is restricted. These regions form loops at the surface of the antibody molecule. These loops provide the binding surface between the antibody and antigen.

Antibodies from non-human animals provoke an immune response to the foreign antibody and its removal from the circulation. Both chimeric and humanised antibodies have reduced antigenicity when injected to a human subject because there is a reduced amount of rodent (i.e. foreign) antibody within the recombinant hybrid antibody, while the human antibody regions do not elicit an immune response. This results in a weaker immune response and a decrease in the clearance of the antibody. This is clearly desirable when using therapeutic antibodies in the treatment of human diseases. Humanised antibodies are designed to have less “foreign” antibody regions and are therefore thought to be less immunogenic than chimeric antibodies.

Various fragments of antibodies are known in the art. A Fab fragment is a multimeric protein consisting of the immunologically active portions of an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, covalently coupled together and capable of specifically binding to an antigen. Fab fragments are generated via proteolytic cleavage (with, for example, papain) of an intact immunoglobulin molecule. A Fab2fragment comprises two joined Fab fragments. When these two fragments are joined by the immunoglobulin hinge region, a F(ab′)2fragment results. An Fv fragment is multimeric protein consisting of the immunologically active portions of an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region covalently coupled together and capable of specifically binding to an antigen. A fragment could also be a single chain polypeptide containing only one light chain variable region, or a fragment thereof that contains the three CDRs of the light chain variable region, without an associated heavy chain variable region, or a fragment thereof containing the three CDRs of the heavy chain variable region, without an associated light chain moiety; and multi specific antibodies formed from antibody fragments, this has for example been described in U.S. Pat. No. 6,248,516. Fv fragments or single region (domain) fragments are typically generated by expression in host cell lines of the relevant identified regions. These and other immunoglobulin or antibody fragments are within the scope of the invention and are described in standard immunology textbooks such as Paul,Fundamental Immunologyor Janeway et al.Immunobiology(cited above). Molecular biology now allows direct synthesis (via expression in cells or chemically) of these fragments, as well as synthesis of combinations thereof. A fragment of an antibody or immunoglobulin can also have bispecific function as described above.

Pharmaceutical Proteins & Peptides

Examples of pharmaceutical proteins include “cytokines”. Cytokines are involved in a number of diverse cellular functions. These include modulation of the immune system, regulation of energy metabolism and control of growth and development. Cytokines mediate their effects via receptors expressed at the cell surface on target cells. Examples of cytokines include the interleukins such as: IL1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 and 33. Other examples include growth hormone, leptin, erythropoietin, prolactin, tumour necrosis factor [TNF] granulocyte colony stimulating factor (GCSF), granulocyte macrophage colony stimulating factor (GMCSF), ciliary neurotrophic factor (CNTF), cardiotrophin-1 (CT-1), leukemia inhibitory factor (LIF) and oncostatin M (OSM), interferon α, interferon β, interferon ε, interferon κ and ω interferon.

Examples of pharmaceutically active peptides include GLP-1, anti-diuretic hormone; oxytocin; gonadotropin releasing hormone, corticotrophin releasing hormone; calcitonin, glucagon, amylin, A-type natriuretic hormone, B-type natriuretic hormone, ghrelin, neuropeptide Y, neuropeptide YY3-36, growth hormone releasing hormone, somatostatin; or homologues or analogues thereof.

The term “chemokine” refers to a group of structurally related low-molecular weight factors secreted by cells having mitogenic, chemotactic or inflammatory activities. They are primarily cationic proteins of 70 to 100 amino acid residues that share four conserved cysteine residues. These proteins can be sorted into two groups based on the spacing of the two amino-terminal cysteines. In the first group, the two cysteines are separated by a single residue (C-x-C), while in the second group they are adjacent (C-C). Examples of members of the ‘C-x-C’ chemokines include but are not limited to platelet factor 4 (PF4), platelet basic protein (PBP), interleukin-8 (IL-8), melanoma growth stimulatory activity protein (MGSA), macrophage inflammatory protein 2 (MIP-2), mouse Mig (m119), chicken 9E3 (or pCEF-4), pig alveolar macrophage chemotactic factors I and II (AMCF-1 and —II), pre-B cell growth stimulating factor (PBSF), and IP10. Examples of members of the ‘C-C’ group include but are not limited to monocyte chemotactic protein 1 (MCP-1), monocyte chemotactic protein 2 (MCP-2), monocyte chemotactic protein 3 (MCP-3), monocyte chemotactic protein 4 (MCP-4), macrophage inflammatory protein 1 α (MIP-1-α), macrophage inflammatory protein 1β (MIP-1-β), macrophage inflammatory protein 1-γ (MIP-1-γ), macrophage inflammatory protein 3 α (MIP-3-α, macrophage inflammatory protein 3 β (MIP-3-β), chemokine (ELC), macrophage inflammatory protein-4 (MIP-4), macrophage inflammatory protein 5 (MIP-5), LD78 β, RANTES, SIS-epsilon (p500), thymus and activation-regulated chemokine (TARC), eotaxin, 1-309, human protein HCC-1/NCC-2, human protein HCC-3.

A number of growth factors have been identified which promote/activate endothelial cells to undergo angiogenesis. These include vascular endothelial growth factor (VEGF A); VEGF B, VEGF C, and VEGF D; transforming growth factor (TGFb); acidic and basic fibroblast growth factor (aFGF and bFGF); and platelet derived growth factor (PDGF). VEGF is an endothelial cell-specific growth factor which has a very specific site of action, namely the promotion of endothelial cell proliferation, migration and differentiation. VEGF is a complex comprising two identical 23 kD polypeptides. VEGF can exist as four distinct polypeptides of different molecular weight, each being derived from an alternatively spliced mRNA. bFGF is a growth factor that functions to stimulate the proliferation of fibroblasts and endothelial cells. bFGF is a single polypeptide chain with a molecular weight of 16.5 Kd. Several molecular forms of bFGF have been discovered which differ in the length at their amino terminal region. However the biological function of the various molecular forms appears to be the same.

