WO1997007216A1

WO1997007216A1 - Deuteriated substances

Info

Publication number: WO1997007216A1
Application number: PCT/GB1996/001988
Authority: WO
Inventors: Jill Barber
Original assignee: The Victoria University Of Manchester
Priority date: 1995-08-19
Filing date: 1996-08-14
Publication date: 1997-02-27
Also published as: AU6748796A; GB2304343A; CA2230051A1; JPH11511023A; AU721877B2; GB9517026D0; EP0845038A1

Abstract

The present invention provides a method for producing a substantially deuteriated substance comprising the steps of growing on a deuteriated medium an organism producing the substance, wherein the organism has been adapted to deuteriated medium such that on a deuteriated medium it produces approximately the same quantity of the deuteriated substance as the non-adapted organism produces of the non-deuteriated substance on a non-deuteriated medium. Also provided are substances produced according to the method of the present invention, MRE600 for use in a method of production of a substantially deuteriated substance, and substantially deuteriated EFTu having a substantially greater half-life in the presence of chymotrypsin in protiated media than protiated EFTu.

Description

DEUTERTATED SUBSTANCES

The present invention concerns methods for making substantially deuteriated substances and substances produced according to the method ofthe present invention.

Fully deuteriated proteins are being used increasingly in both NMR (Nuclear Magnetic Resonance) and neutron scattering experiments. Deuteriation increases the resolution obtainable in neutron scattering, and allows proteins under study (deuteriated) to be distinguished from background (natural abundance) (Moore, P., 1979, Methods in Enzymology, 59: 639-655). In Η NMR studies of ligand-protein interactions, the protein can be rendered transparent by deuteriation (Seeholzer, S.H., et al., 1986, Proc. Natl. Acad. Sci. USA., 83: 3634). 'H-'H dipole-dipole interactions are reduced resulting in relatively narrow signals in one- and multi-dimensional spectra, and the quality of NOES Y spectra is further improved by the reduction in spin diffusion effects (Brennan, R.J., et al, 1994, J. Chem. Soc. Chem. Commun., 1615).

Deuteriated proteins are normally obtained by growing E. coli bearing a suitable overproducing vector on a fully deuteriated medium. The best medium used to date is deuteriated algal hydrolysate, but this is difficult to prepare, expensive and subject to fluctuations in availability (Daboll, H.F., et al, 1962, Biotechnology and Bioengineering, IV: 281). The most widely used alternative is based on d₄-succinate and D₂0, but this medium is generally characterised by poor yields of protein (e.g. Oda, Y., et al, 1992, J. Biomol. NMR, 2: 137-147). Good yields of deuteriated chloramphenicol acetyltransferase have been obtained using d₄-succinate medium, but even these were a factor of four lower than yields of natural abundance protein prepared using the same clone (Derrick. J.P., et al, 1992. Biochemistry, 31: 8191-8195). The present invention overcomes the disadvantages of the prior art and provides a simple, convenient, efficient, and less expensive method for producing substantially deuteriated substances.

According to the present invention there is provided a method for producing a substantially deuteriated substance comprising the steps of growing on a deuteriated medium an organism producing the substance, wherein the organism has been adapted to deuteriated medium such that on a deuteriated medium it produces approximately the same quantity of the deuteriated substance as the non-adapted organism produces ofthe non-deuteriated substance on a non-deuteriated medium.

The organism may be unicellular or multicellular. The organism may, for example, be a bacterium, a prokaryote, an alga or a fungus.

The substance may for example be a protein, an antibiotic or nucleic acid. The substance may be the bacterial elongation factor Tu (EFTu).

The substance produced may be at least 85% deuteriated. It may for example be at least 90% or 95% deuteriated.

The organism may have been adapted by growth in a series of cultures, the first culture containing less deuterium than the final culture.

The organism may have been grown to an A₅₅₀ of approximately 1.0 in each ofthe series of cultures.

Each culture in the series may contain more deuterium than the preceding culture. The cultures may contain d₄-succinate and D₂0 as the sources of deuterium.

The series may be a series of at least three cultures. It may be a series of 3, 4, 5, 6, 7, 8, 9 or 10 cultures.

The organism may have been adapted by growth in a series of cultures containing d₄-succinate and D₂0, each culture ofthe series containing more deuterium than the preceding culture. It may for example have been a series of four cultures containing 70%, 80%, 90% and 100% D₂0 respectively.

The organism may also have been further adapted to growth at a raised temperature. It may have been adapted to growth at 42°C. By 'raised temperature' is meant a raised temperature relative to the normal growth temperature for the organism, in the case of bacteria this being about 37°C.

The substance may be subsequently harvested.

The adapted organism may be E. coli or a derivative thereof. It may be derived from E. coli MRE600. It may for example be Bacillus subtilis or streptomyces, e.g. Saccharopolyspora erythraea. Derivatives of a micro-organism may be progeny of the micro-organism in which it has e.g. given up DNA to, or accepted DNA from, another micro-organism, the progeny exhibiting the same, or substantially the same, characteristics.

The adapted organism may be E. coli MRE600D. The adapted organism may have been transformed. It may have been transformed with the pJBDB02 vector (also known as pTrc99AtufA, the tufA gene encoding EFTu).

The adapted organism may produce EFTu or a fragment, for example an immunogenic fragment, or a partially modified form thereof. Partial modification may for example be by way of addition, deletion or substitution of amino acid residues. A substitution may for example be a conserved substitution. Hence a partially modified molecule may be a homologue ofthe molecule from which it was derived. It may for example have at least 40%, for example 50, 60, 70, 80, 90 or 95% homology with the molecule from which it is derived.

The adapted organism may be E. coli MRE600DpJBDB02.

Also provided according to the present invention are substances produced according to the method ofthe present invention.

Bacterial elongation factor Tu is a 43 kDa protein which plays a crucial role in protein synthesis. It is the most extensively studied member of the G binding protein family, its role in bacteria being to deliver aminoacyl tRNA to the ribosome. EFTu binds a number of ligands including GTP, GDP, EFTs, aminoacyl tRNA, the ribosome, the antibiotics kirromycin and pulvomycin, and DNA. EFTu is too large for its interactions with these ligands to be studied satisfactorily with NMR using naturally abundant EFTu. However, deuteriated EFTu's interactions with these ligands may be readily studied, revealing hitherto unknown information about EFTu's interactions.

According to the present invention there is also provided MRE600 or a derivative thereof for use in the production of a substantially deuteriated substance. The MRE600 may be for use in a method according to any one ofthe preceding claims. Also provided according to the present invention is substantially deuteriated EFTu having a substantially longer half-life in protiated media in the presence of chymotrypsin than protiated EFTu.

Using the method of the present invention (see 'Experimental' section below), substantially deuteriated EFTu has been prepared, allowing it to be studied with NMR.

The invention will be further apparent from the following description which shows, by way of example only, one form of method of producing substantially deuteriated substances. Ofthe figures:

Figure 1 shows plasmid pJBDB02. Bases 1512-270 (the tufA EcoRI restriction site to the Smal restriction site) are derived from pTrc99A and the J226 bp tufA fragment was isolated from pJBDBOl by EcoRI digestion;

Figure 2 shows SDS PAGE of crude cell lysate from MRE600DpJBDB02 cells grown on deuteriated succinate medium. Lanes 1 and 6 contain molecular weight markers of 14, 20, 30, 43, 67 and 94 kDa. Lane 2 contains a sample of crude cell lysate from a non- induced culture; lane 3 contains a sample of crude cell lysate from am IPTG-induced culture; lanes 4 and 5 contain 1 :10 dilutions of the lysate samples loaded into lanes 2 and 3 respectively. The strong band of EFTu at 43 kDa is clearly visible in samples from the induced culture and the high yield of EFTu in relation to other cell proteins is demonstrated;

Figure 3 shows the degradation of perdeuteriated and protiated EFTu at 42 °C in H₂0 (pH 8.0) and D₂0 (pD 8.0); Figure 4 shows the degradation of perdeuteriated and protiated EFTu at 51 °C in D₂0 (pD 8.0);

Figure 5 shows the degradation by chymotrypsin of protiated and perdeuteriated EFTu in protiated media; and

Figure 6 shows the degradation by chymotrypsin of protiated and perdeuteriated EFTu in deuteriated media.

EXPERIMENTAL

Bacteria were adapted to growth on deuteriated succinate medium and were transformed with a plasmid DNA vector. Resultant clones were screened for the presence ofthe vector and their ability to synthesise deuteriated EFTu.

Results show that the method of the present invention has allowed the preparation of very high yields (nearly 100 mg/litre of culture medium) of deuteriated EFTu using a series of cultures containing d₄-succinate/D₂0 medium. The yields are equal to those obtained using natural abundance medium and the same clone. These results compare extremely well with the prior art results which showed a four-fold reduction in protein production levels when bacteria were cultured on deuteriated medium (Derrick et al, 1992).

Synthesis of deuteriated sodium succinate

Deuteriated sodium succinate was prepared essentially as described by Stella, V.J., et al. (1973, J. Pharm. Sci., 62: 634) and LeMaster, D.M. and Richards, F.M. (1981, Analytical Biochemistry, 122: 238-247).

Adaptation ofMRE600 to growth on deuteriated succinate medium Deuteriated sodium succinate medium was prepared as follows: 0.1 g NaOH, 2.4 g KH₂P0₄, 1.1 g deuteriated sodium succinate, 0.2 g (NH₄)₂S0₄, 30 mg FeS0₄, 1 μl cone. H₂S0₄ (Fissons) were slurried in 1 ml of ²H₂0 (D₂0; Fluorochem) and stirred for 1 hour. The mixture was freeze dried and resuspended in 100 ml of ²H₂0 and filter sterilised. For preparation of agar plates, 1.5 g bacto agar were added to 100 ml medium which was sterilised by autoclaving at 15 lb/sq.inch (approx. 10546 kg/πr)for 20 minutes at 121 °C, in a sealed Duran bottle. E. C0/ MRE6OO (National Collection of Industrial and Marine Bacteria) was adapted to growth on deuteriated medium by growing successive liquid cultures in medium containing deuteriated sodium succinate as a carbon source in 70%, 80%, 90% and, finally, 100% ²H₂0. The cells were then plated on 100% ²H₂0 deuteriated succinate agar plates and incubated for 48 hours at 37°C at which time a single colony was selected and grown in liquid culture until stationary phase was reached. Glycerol stocks were made from the culture, and the adapted cells were stored at -20 °C for periods of up to 1 year. MRE600 adapted in this way are denoted MRE600D.

Construction ofthepJBD02 vector

A 1226 bp fragment encoding the tufA was excised from pJBDBOl (Bloor, D.J. and

Barber, J., 1993, Biochemistry and Molecular Biology International, 31(4): 727-731) by

EcoRI digestion. The fragment was isolated from a low melting point agarose gel (BRL;

Ultrapure RTM) using Geneclean (Stratech; RTM), and ligated into the EcoRJ site of pTrc99A. The ligation products were used to transform competent MRE600D cells (see below).

Transformation ofMRE600D with Plasmid DNA

E. coli MRE600D cells grown for a single generation on Luria's broth (LB) medium were made competent by treatment with calcium chloride and were transformed according to the method of Mandel, M. and Higa, A. (1970, J. Mol. Biol., 53_: 154-162). 10 μl of ligation products containing 100 ng of Trc99A DNA were added to 200 μl of competent cells and the suspension was incubated on ice for 1-3 hours before heat shocking at 42 °C for 2 minutes. LB medium was used in the transformation procedure, and transformants were placed on LB agar plates supplemented with 100 μg/ml ampicillin (Sigma). Following incubation overnight at 37°C, colonies were replica plated onto deuteriated succinate agar plates. Screening of MRE600DpJBDB02 clones

10 transformants were grown in LB medium and small scale plasmid isolations were carried out using the Promega Magic Miniprep (RTM) system. Plasmids were screened by Smal digestion and agarose gel electrophoresis, and plasmids shown to contain the tufA gene in the correct orientation with respect to the trc promoter of pTRC99A were denoted pJBDB02 (see Figure 1). A single MRE600DρJBDB02 clone was then picked from the deuteriated succinate replica plate and used to inoculate 40 ml of deuteriated succinate medium containing 100 μg/ml ampicillin. The culture was grown at 37°C with shaking 250 rpm (³/4 inch (1.9 cm) stroke). After approximately 40 hours incubation, when the optical density measured at 550 nm (OD₅₅₀) ofthe culture had reached 0.4, the culture was divided between two sterile 250 ml flasks. One ofthe cultures was induced by the addition of isopropylthiogalactoside (IPTG; NovaBiochem) to a final concentration of 1 mM, and incubation of both was continued as described. After a further 24 and 48 hours growth, two 1 ml samples were taken from each flask_r and the cells isolated and lysed as described previously (Bloor and Barber, 1993, supra). Measurement of EFTu levels in the crude cell lysates was carried out using the GDP exchange assay described previously by Arai, K. et al (1980, Proc. Natl. Acad. Sci. USA., 77(3): 1326-1330). SDS PAGE was also used to evaluate EFTu expression by both the induced and non-induced cultures (Pharmacia Phastgel System), and total cell protein in crude cell lysates was estimated using the Bradford Assay (Bradford, M.M., 1976, Analytical Biochemistry, 72: 248-254).

RESULTS AND DISCUSSION

E. coli MRE600 which had not undergone adaptation to deuteriation could not be cultured in deuteriated medium. E. coli MRE600 which had undergone adaptation achieved a maximum OD₅₅₀ of 6.0 and cultures had a doubling time of approximately 6 hours. Plating deuterium adapted bacteria on deuteriated agar plates proved to be particularly important, resulting in improved growth rates and much reduced variability between cultures.

Competent cells prepared from MRE600D cells grown for a single generation on protiated LB medium were successfully transformed with plasmid pBR322 when large quantities (100 ng) of DNA were used. Using the same procedure, MRE600D cells were also successfully transformed with plasmid pJBDB02 and, following transformation, it was possible to isolate transformants which had retained their ability to grow on deuteriated medium from replica deuteriated succinate agar plates. The feasibility of storing bacteria grown on deuteriated medium as frozen glycerol stocks has also been demonstrated.

Measurement of EFTu levels in crude lysates of MRE600DpJBDB02 grown on deuteriated medium showed that the IPTG-induced culture produced an excellent 100 mg of labelled EFTu per litre of cell culture. Non-induced culture of MRE600DpJBDB02 cells produced only 12 mg of EFTu per litre of culture. Figure 2 shows the result of SDS PAGE of a samples crude lysate obtained from induced and non- induced MRE60DpJBDB02 cells grown on deuteriated media.

The yield of EFTu from deuteriated cultures ofthe MRE600DpJBDB02 clone was more than four times higher than that obtained from deuteriated cultures of MRE600pCP40pCI857 (Howard, T.D., 1991, PhD Thesis. University of Manchester, UK; Kennedy, K.K.. 1992, PhD Thesis, University of Manchester, UK). Expression of tufA in the MRE600pCP40pCI857 clone (which had previously been the best overproducer of deuteriated EFTu available), is under the influence of a λ_PL promoter, but there is some evidence to suggest that the EcoRl/Hindlll fragment encoding tufA in pCP40 also encodes regulatory sequences which may influence expression ofthe gene under conditions of poor growth (van der Meide, R.A. et al, 1983, European Journal of Biochemistry, 130: 409-417).

Additional EFTu was isolated using the method of Wittinghoefer, A. and Leberman, R., 1976, Eur. J. Biochem., 62: 373-382. Electrospray mass spectrometry of the perdeuteriated protein gave a molecular ion of 45574 which fell to 45527 on 12 hours incubation under mildly denaturing conditions (20% H₂0, 1% HCOOH in CH₃CN). Under the same conditions, with D₂0 replacing H₂0, the molecular weight ofthe normal protiated protein rose from 43219 to 43860 corresponding to the exchange of 637 hydrogens. The EFTu molecule contains 668 hydrogens linked to heteroatoms, so these data indicate that about 30 of these do not exchange under these conditions. Taking this into account, these results indicate that the deuteriated protein was 95% deuteriated at the CD positions, and that about 78 (12%) of the deuteriums attached to heteroatoms survived the long (8 hours) work up in H₂0.

Additionally, solutions of protiated or perdeuteriated EFTu were prepared at 0.5-0.6 mg ml^"1 in 64.4 mM Tris buffer, pH 8 in H₂0 or pD 8 in D₂0. Samples of each ofthe four solutions (protiated EFTu in H₂O and D₂0, perdeuteriated EFTu in H₂0 and D₂0 were allowed to denature at 8 °C, room temperature at 42 ^CC (Figure 4). Protiated and perdeuteriated EFTu in D₂0 were also degraded at 51 °C. All experiments were carried out in duplicate. Samples were removed at intervals and assayed for EFTu by the standard GDP exchange assay.

It is clear from the results (Figures 3 and 4; Table 1) that D₂0 has a stabilizing effect on both protiated and perdeuteriated EFTu at all the temperatures measured. For example at 42 °C the half life of normal EFTu is 70 minutes in H₂O but 350 minutes in D₂0. D₂0 is known to affect the stability of proteins in aqueous solution. In many cases the effect is stabilizing, but destabilizing effects have also been reported.

The denaturation of EFTu followed complex kinetics, approaching a normal exponential decay only at elevated temperature (42 °C for H₂0 solutions, 51 °C for D₂0 solutions). At elevated temperatures the errors are negligible whereas the slow initial phase of degradation seen at lower temperatures varies slightly in duration (see Table 1). It can be seen, however, that perdeuteriation ofthe protein is under almost all conditions stabilizing.

The residual heteroatom deuteriums, which are presumably buried in the core ofthe protein, are expected to be stabilizing as deuterium bonds are stronger than hydrogen bonds. The destabilizing effect therefore presumably derives from the.carbon- bound deuteriums (of which there are 2399). EFTu is an unstable protein which associates readily and which is stabilized in concentration solution. The destabilization of EFTu on perdeuteriation appears to arise from changes in hydrophobic interactions, including a decrease in association behaviour.

Experiments have also shown (Figures 5 and 6) that perdeuteriated EFTu has a significantly longer half-life (60 minutes compared to 40 minutes) in the presence of chymotrypsin than protiated EFTu in protiated media. TABLE 1

Half-lives of protiated and perdeuteriated EFTu in H₂0 and D₂0 at various temperatures

Temperature (°C)

8 19 42 52

Protiated EFTu in H₂0 t, (mins) a 1400±100 67.5±12.5 -

Protiated EFTu in D₂0 t_Λ (mins) a >3300 350±30 30±1

Deuteriated EFTu in H₂0 t._Λ (mins) a 1750±50 70±2 -

Deuteriated EFTu in D₂0 t,_Λ (mins) a >2000 225±25 lO±l

a - does not degrade appreciably after 55 hours

t_/2 represents the time after which one half of the protein has degraded. This is a true half life only at elevated temperatures.

Claims

1. A method for producing a substantially deuteriated substance comprising the steps of growing on a deuteriated medium an organism producing the substance, wherein the organism has been adapted to deuteriated medium such that on a deuteriated medium it produces approximately the same quantity ofthe deuteriated substance as the non-adapted organism produces ofthe non-deuteriated substance on a non-deuteriated medium.

2. A method according to claim 1 wherein the organism is unicellular.

3. A method according to claim 1 wherein the organism is multicellular.

4. A method according to any one of claims 1-3 wherein the organism is selected from any one ofthe group of classes of bacterium, prokaryote, alga and fungus.

5. A method according to claim 1 wherein the substance is either a protein or an antibiotic.

6. A method according to claim 5 wherein the substance is the bacterial elongation factor Tu.

7. A method according to any one of the preceding claims wherein the substance produced is at least 85% deuteriated.

8. A method according to claim 7 wherein the substance produced is at least 90% deuteriated.

9. A method according to claim 8 wherein the substance produced is at least 95% deuteriated.

10. A method according to any one of the preceding claims wherein the organism has been adapted by growth in a series of cultures, the first culture containing less deuterium than the final culture.

11. A method according to claim 10 wherein the organism has been grown to an A₅₅₀ of approximately 1.0 in each ofthe series of cultures.

12. A method according to either one of claims 10 or 11 wherein each culture contains more deuterium than the preceding culture.

13. A method according to any one of claims 10-12 wherein the .cultures contain d₄-succinate and D₂0.

14. A method according to any one of claims 10-13 wherein the series is a series of at least three cultures.

15. A method according to claim 14 wherein the series is a series selected from any one ofthe group of 3, 4, 5, 6, 7, 8, 9, and 10 cultures.

16. A method according to any one of claims 10-15 wherein the organism has been adapted by growth in a series of cultures containing d₄-succinate and D₂0, each culture ofthe series containing more deuterium than the preceding culture.

17. A method according to claim 16 wherein it is a series of four cultures containing 70%, 80%, 90% and 100% D₂0 respectively.

18. A method according to any one of the preceding claims wherein the organism is further adapted to growth at a raised temperature.

19. A method according to claim 18 wherein the organism is adapted to growth at 42° C.

20. A method according to any one of the preceding claims wherein the substance is subsequently harvested.

21. A method according to any one of the preceding claims wherein the adapted organism is E. coli or a derivative thereof.

22. A method according to claim 21 wherein the adapted organism is derived from£. cø// MRE600.

23. A method according to claim 22 wherein the adapted organism is E. coli MRE600D.

24. A method according to any one of the preceding claims wherein the adapted organism has been transformed.

25. A method according to claim 21 wherein the adapted organism has been transformed with the pJBDB02 vector.

26. A method according to any one of claims 18-22 wherein the adapted organism produces EFTu or a fragment thereof.

27. A method according to any one of claims 18-23 wherein the adapted organism is E. coli MRE600DpJBDB02.

28. A substance produced according to the method of any one ofthe preceding claims.

29. MRE600 for use in a method of production of a substantially deuteriated substance.

30. MRE600 according to claim 29 for use in a method according to any one of claims 1-27.

31. Substantially deuteriated EFTu having a substantially greater half-life in the presence of chymotrypsin in protiated media than protiated EFTu.