WO2004056850A2

WO2004056850A2 - Mutant proteins showing increased secretion

Info

Publication number: WO2004056850A2
Application number: PCT/EP2003/051050
Authority: WO
Inventors: Lothar Steidler; Sabine Neirynck
Original assignee: Vib Vzw; Universiteit Gent
Priority date: 2002-12-19
Filing date: 2003-12-18
Publication date: 2004-07-08
Also published as: AU2003303222A8; WO2004056850A3; AU2003303222A1

Abstract

The present invention relates to proteins that carry a mutation in the amino terminal part of the mature protein, which causes an improved secretion of the mutated protein form in gram-positive bacteria, without affecting the biological activity. More particularly, it relates to a protein where a proline has been replaced in the first (10) amino acids of the mature protein. In a preferred embodiment, the invention relates to hypersecreting mutants of interleukin (6) and interleukin (10).

Description

LST/ALA/V135

MUTANT PROTEINS WITH INCREASED SECRETION

The present invention relates to proteins that carry a mutation in the amino terminal part of the mature protein, which causes an improved secretion of the mutated protein form in gram-positive bacteria, without affecting the biological activity. More particularly, it relates to a protein where a proline has been replaced in the first 10 amino acids of the mature protein. In a preferred embodiment, the invention relates to hypersecreting mutants of interleukin 6 and interleukin 10.

Protein secretion of heterologous proteins by recombinant microorganisms has been an important topic in biotechnology. In many cases, protein secretion is highly wanted to improve yield and to facilitate downstream processing. Indeed, if the heterologous protein is not secreted in an efficient way, lysis of the host may be needed, which will contaminate the heterologous protein with indigenous proteins and complicate the purification. A lot of work on protein secretion has been carried out in the gram-negative bacterium Escherichia coli. The choice for E. coli has rather been historical than practical, as it is difficult to reach a good secretion in this organism, and heterologous proteins are often found in inclusion bodies within the cell. During the process of cell lysis and solubilization, the heterologous protein may often lose its biological activity, due to irreversible denaturation. Several alternative host organisms have been tried to improve secretion. Several proteins are produced in Saccharomyces cerevisiae, but this yeast has also only a limited capacity for secretion. Other yeasts, such as Pichia spp. may be better in that respect. However, although they may have certain advantages, yeasts are in general more fastidious than bacteria, and the cell density of the culture may be lower. Gram-positive bacteria, such as Bacillus or lactic acid bacteria do form an interesting alternative. Protein secretion in Bacillus is easier than in E. coli, but an important drawback is the protease production by these organisms, resulting in a possible breakdown of the proteins produced. Lactic acid bacteria, such as Lactobacillus or Lactococcus spp. may form an interesting alternative, especially because these organisms may be used for delivery of biological active molecules in vivo, whereby secretion of the biological active molecule is essential. Delivery of biological active molecules by recombinant microorganisms is know by the person skilled in the art, and have been disclosed, amongst others, in W09611277 and WO9714806. LST/A A V135

Although it is generally accepted that the sequence of the mature protein is not important for secretion, we have found that proteins, carrying a proline in the first 10 amino acids of the mature form may be hampered in their secretion, even in gram- positive bacteria. Surprisingly, we noted that replacement of this proline by another amino acid is dramatically improving the secretion in gram-positive bacteria.

A first aspect of the invention is a mutant protein showing improved secretion in a gram-positive bacterium, whereby one or more proline residues within the first 10 amino acids of the mature protein have been replaced by another amino acid. Preferably, the first praline within these first 10 amino acids has been replaced. Even more preferably, all proline residues out of those first 10 amino acids of the mature protein have been replaced by another amino acid. Preferably, said other amino acid is an alanine. Even more preferably, said replacement is not significantly affecting the biological activity of said mature protein. One preferred embodiment is a mutant interleukin 10 protein according to the invention. Preferably, said interleukin 10 comprises SEQ ID N° 8, even more preferably, said interleukin 10 is essentially consisting of SEQ ID N° 8, most preferably, said interleukin 10 consists of SEQ ID N° 8. Another preferred embodiment is a mutant interleukin 6 protein according to the invention. Preferably, said interleukin 6 comprises SEQ ID N° 10, even more preferably, said interleukin 6 is essentially consisting of SEQ ID N° 10, most preferably, said interleukin 6 consists of SEQ ID N° 10.

Preferably, said gram-positive bacterium is a lactic acid bacterium, even more preferably, it is selected from the group consisting of Lactococcus lactis, Lactobacillus salivarius and Lactobacillus acidophilus. Another aspect of the invention is a nucleic acid, encoding a mutant protein according to the invention.

Still another aspect of the invention, is an expression vector for gene expression on a gram-positive bacterium, comprising a nucleic acid according to the invention. Preferably, said gram-positive bacterium is a lactic acid bacterium, even more preferably, it is selected from the group consisting of Lactococcus lactis, Lactobacillus salivarius and Lactobacillus acidophilus.

A further aspect of the invention is the use of the replacement of one or more proline residues, occurring in the first 10 amino acids of a mature protein, by another amino acid, to obtain improved secretion in a gram-positive bacterium. Preferably, the first proline within these first 10 amino acids has to be replaced. Even more preferably, all LST/ALA/V135

proline residues occurring in the first 10 amino acids have to be replaced. Even more preferably, said other amino acid is an alanine. Most preferably, said replacement is not significantly affecting the biological activity of the mature protein. Preferably, said gram-positive bacterium is a lactic acid bacterium, even more preferably, it is selected from the group consisting of Lactococcus lactis, Lactobacillus salivarius and Lactobacillus acidophilus.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Construction of pT1hlL10PxA and pT1hlL10ApxA. The names hlL10s, hlLIOa, hlLIOSpxA, HILIOADPxA and 01hlL10 to 28hlL10 refer to the primers used; the sequence of the primers is listed in the sequence listing.

Figure 2: Construction of pT1plL6 and pT1plL6m. The names plL6s, plL6a, plL6mut and plL6-1 to plL6-8 refer to the primers used. The sequence of the primers is listed in the sequence listing Figure 3: western blot of L. lactis MG1363 containing control plasmid pTREX (lanes 1 , 2), pT1hlL10 (lanes 3, 4), pT1hlL10PxA (lanes 5, 6), pT1hlL10A (lanes 7, 8) or pT1hlL10APxA (lanes 9, 10). Odd numbers show the equivalent of 1 ml culture supernatant and even numbers the equivalent of 150 μl cell fraction. The precursor protein (p) is present in the cell fractions of all hlL10 constructs, while the mature hlL10 is detected in the growth medium.

Figure 4: Amount of hlL10 in the culture supernatant as determined in a sandwich ELISA.

Figure 5: western blot of L. lactis MG1363 containing control plasmid pT1NX (lanes 1, 2), pT1plL6m15 (lanes 3, 4), pT1plL6m24 (lanes 5, 6) or pT1plL6 (lanes 7, 8). Odd numbers show the equivalent of 1 ml culture supernatant and even numbers the equivalent of 150 μl cell fraction. The precursor protein (p) is present in the cell fractions of all plL6 constructs, while the mature plL6 can only be detected in the growth medium of the mutated plL6 constructs. LST/ALA/V135

EXAMPLES

Materials and methods to the examples

Unless otherwise stated, PCR amplification of DNA was performed with VENT polymerase and using conditions recommended by the manufacturer. Restriction endonucleases were used under standard conditions and in buffers recommended by the manufacturers. General molecular cloning techniques and electrophoresis of DNA and proteins were carried out essentially as described (Sambrook et al., 1990). L. lactis was transformed by electroporation of cells grown in glycine (Wells et al., 1993).

Throughout the study, L lactis MG1363 (Gason, 1983) was used. Precultures of L lactis MG1363 were grown in M17 (Difco, St. Louis) supplemented with 0.5% w/v of glucose (GM17). Cultures were carried out in BM9. BM9 contains per liter 6g of Na₂HP0₄, 3g of KH₂P0₄, 1g of NH₄CI, 0.5g of NaCI, 2 mmol of MgS0₄, 25 mmol of NaHC0₃, 25 mmol of Na₂C0 , 0.1 mmol of CaCI₂, 5g of glucose and 5 g of casitone (Difco). Where appropriate, the medium is supplemented with 5μg/ml of erythromycin.

Example 1: replacement of proline on position 2 of human Interleukin 10 by alanine results in an improved secretion in Lactic acid bacteria.

The mature human IL10 (genbank M57626, 85 - 564, SEQ ID N° 1) was cloned in pTINX, after the usp45 secretion leader (Van Asseldonk et al., 1990), resulting in pT1hlL10. pTINX is a pTREXI (Wells and Schofield, 1996) derivative; the construction of pTINX has been described in WO0023471. The construction of the plasmid is summarized in Figure 1.

With pT1hlL10 only a small amount hlL10 was obtained in the growth medium. When the second codon of the mature protein, proline, was mutated to an alanine (pT1hlL10PxA, mutated form represented by SEQ ID N° 3), a better secretion of hlL10 in the culture supernatant was obtained. By adapting the codon usage of hlL10 to fit the codon usage in Lactococcus lactis (as presented in SEQ ID N° 5; incorporated in plasmid pT1hlL10A), a higher concentration of hlL10 in the growth medium could also be obtained. The highest expression level however was obtained when the mutation, proline to alanine and the adapted codon usage were combined (pT1hlL10APxA, sequence of the mutated form represented in SEQ ID N° 7). An overview of the expression level with the different constructs is shown in Figure 3 and 4. LST/ALAΛ/135

Example2: replacement of the proline on position 1 of porcine Interleukin 6 by alanine results in an improved secretion in Lacϋc acid bacteria.

The mature porcine IL6 (genbank M80258, 147-698) was cloned in pTINX, after the usp45 secretion leader. Therefore, a partial plL6 fragment was amplified from pBLUESILβ (gift from Prof. E. Cox), and cloned in pUC19, resulting in pUCplL6-1. The missing 3' part was synthesized according to the method of Stemmer et al. (1995) and cloned in pUCplL6. With this construct (pT1plL6). The resulting plasmid, pUCplL6-2 contained the complete cDNA sequence of plL6. From this plasmid, the sequence of mature IL6 was isolated and cloned into pTINX. The sequence of this construct, carrying the natural proline as first amino acid of the mature form, showed to be correct, but no secretion of plL6 was detected in the growth medium when this construct was transformed into Lactococcus lactis MG1363. However, when the first codon of the mature protein was mutated from proline to an alanine (resulting in pT1plL6m), a good expression of plL6 in the culture supernatant was obtained. An overview of the constructions is shown in Figure 2. The results of the expression experiments are shown in Figure 5. pT1plL6m15 and pT1plL6m24 are two independent isolates of the same construct, and do not differ from each other. Determination of the N-terminus showed correct processing of the precursor. The secretion in the medium was verified using a Western blot, with polyclonal rabbit anti pig IL6 (Endogen, Wobum, MA, USA) at a 1/500 dilution as first antibody, and polyclonal goat anti-rabbit IgG-AP (SBA, Birmingham, AL, USA) at a 1/1000 dilution as secondary antibody.

LST/ALA/V135

REFERENCES

- Gasson, M.J. (1983). Plasmid complements of Streptococcus lacϋs NCDO 712 and other lactic streptococci after protoplast-induced curing. J Bacteriol 154,1-9.

- Sambrook, J., Fritsch, E.F. and Maniatis, T. (1990) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, New York

- Stemmer, W.P., Crameri, A., Ha, K.D., Brennan, T.M. and Heyneker, H.L. (1995). Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. Gene 164, 49-53.

- Van Asseldonk, M., Rutten, G., Oteman, M., Siezen, R.J., de Vos, W.M. and Simons, G. (1990). Cloning of usp45, a gene encoding a secreted protein from

Lactococcus lactis subsp. lactis MG1363. Gene 95, 155-160.

- Wells, J.M. and Schofield: in Lactic Acid Bacteria: current advances in metabolism, genetics and applications. F. Bozoglu and R. Bibek, eds., Nato ASI Series H, Vol. 98, p. 37, Springer Verlag, 1996. - Wells, J.M., Wilson, P.W. and Le Page, R.W.F. (1993). Improved cloning vectors and transformation procedure for Lactococcus lactis. J Appl Bacteriol 74, 629-636.

Claims

LST/ALA/V135CLAIMS

I. A mutant protein showing improved secretion in a gram-positive bacterium, whereby one or more proline residues within the first 10 amino acids of the mature protein have been replaced by another amino acid.

2. A mutant protein according to claim 1 , whereby all proline residues within the first 10 amino acids have been replaced by another amino acid.

3. A mutant protein according to claim 1 or 2, whereby said other amino acid is an alanine.

4. A mutant protein according to any of the claims 1-3, whereby said mutant protein is interleukin 10.

5. A mutant protein according to claim 4, whereby said interleukin 10 comprises SEQ ID 8.

6. A mutant protein according to any of the claims 1-3, whereby said mutant protein is interleukin 6.

7. A mutant protein according to claim 6, whereby said interleukin 6 comprises SEQ ID 10.

8. A mutant protein according to any of the previous claims, whereby said gram- positive bacterium is a lactic acid bacterium.

9. A mutant protein according to claim 8, whereby said lactic acid bacterium is selected from the group consisting of Lactococcus lactis, Lactobacillus salivarius and Lactobacillus acidophilus.

10. A nucleic acid, encoding a mutant protein according to any of the previous claims.

II . An expression vector for gene expression in a gram-positive bacterium, comprising a nucleic acid according to claim 10.

12. An expression vector according to claim 11, whereby said gram-positive bacterium is a lactic acid bacterium.

13. An expression vector according to claim 12, whereby said lactic acid bacterium is selected from the group consisting of Lactococcus lactis, Lactobacillus salivarius and Lactobacillus acidophilus.

14. The use of the replacement of one or more proline residues, occurring in the first 10 amino acids of a mature protein, by another amino acid, to obtain improved secretion in a gram-positive bacterium.

15. The use according to claim 14, whereby all proline residues occurring in the first 10 amino acids have been replaced. LST/ALAΛ 135

16. The use according to claim 14 or 15, whereby said other amino acid is an alanine.

17. The use according to any of the claims 14-16, whereby said gram-positive bacterium is a lactic acid bacterium.

18. The use according to claim 17, whereby said lactic acid bacterium is selected from the group consisting of Lactococcus lactis, Lactobacillus salivarius and

Lactobacillus acidophilus.