AUTO-INDUCIBLE EXPRESSION SYSTEM

Abstract

A method for the expression of a protein of interest by a bacterium, notable in that it comprises the culturing of a bacterium temporarily or continuously expressing an Hsp protein, in that said bacterium also comprises a nucleic acid sequence, encoding a protein of interest, under the control of a lac promoter and in that said bacterium is cultured in a medium which does not contain IPTG or a metabolized molecule in such a way as to automatically induce the induction of transcription from the lac promoter.

Claims

1-13. (canceled)

14. A method for the expression of a protein of interest by a bacterium comprising: it comprises the culturing of a bacterium temporarily or continuously expressing an Hsp protein, in that said bacterium further comprises a nucleic acid sequence, encoding a protein of interest, under the control of a lac promoter and in that said bacterium is cultured in a medium not containing IPTG or a metabolised molecule in such a way as to automatically induce the induction of transcription from the lac promoter.

15. The method according to claim 14, wherein said nucleic acid encoding a protein of interest is comprised in a first plasmid.

16. The method according claim 14, wherein said protein of interest is a recombinant protein.

17. The method according to claim 14, wherein said protein of interest is a polymerase.

18. The method according to claim 14, wherein said polymerase is a T7 polymerase.

19. The method according to claim 17, wherein said bacterium further comprises a second nucleic acid sequence, encoding a second protein of interest placed under the control of a specific promoter of said polymerase.

20. The method according to claim 14, wherein said second nucleic acid sequence is comprised in said first plasmid or in a second plasmid.

21. The method according to claim 14, wherein said Hsp protein is encoded by a nucleic acid sequence under the control of a lac promoter.

22. The method according to claim 14, wherein said bacterium is an E. coli.

23. The method according to claim 22, wherein said bacterium belongs to the BL21(DE3) strain or to the BL21(DE3) star strain.

24. The method according to claim 14, wherein the Hsp protein is chosen in the group comprising Hsp40, Hsp70, Hsp90 and Hsp110.

25. The method according to claim 14, wherein the Hsp protein is human Hsp70 protein.

26. The method according to claim 14, wherein said bacterium is cultured in a liquid medium.

Description

DESCRIPTION OF EMBODIMENTS

Expression of Heterologous Proteins by a Bacterium Expressing Human Hsp70

Materials and Methods

[0033] In all the experiments hereinafter, an E. coli BL21(DE3) star bacterial strain was used. However, the invention is not restricted to this particular type of bacteria.

[0034] The sequence encoding human Hsp70 was cloned in the bacterial expression plasmid pET21d and placed under the control of the T71ac promoter. This plasmid further contains an ampicillin resistance gene.

[0035] The plasmid pET21d-Hsp70 was used to convert E. coli BL21(DE3)Star and obtain a bacterium capable of expressing Hsp70.

[0036] A second pET plasmid comprising a kanamycin resistance gene and the same origin of replication as the plasmid pET21d was used to clone a gene encoding a heterologous protein. This second plasmid was used to convert the bacterial strain encoding Hsp70.

[0037] The production of five different heterologous proteins was tested, these proteins are: [0038] Methionine sulphoxide reductase B (MsrB) from Xanthomonas compestris. [0039] Thioredoxin 1 (Trx1) from E. coli. [0040] Purine nucleoside phosphorylase (PNP) from E. coli. [0041] The N-terminal domain of the T1R3 receptor from Homo sapiens. [0042] Miraculin from Richardella dulcifica (MCL).

[0043] These five proteins are from different sources (bacterium, plant or human) and of sizes varying from 14 kDa to 45 kDa. Moreover, they exhibit different functions (enzyme, receptor or ligand) and cellular locations (cytoplasm, plasma or membrane).

[0044] The converted bacteria were cultured in LB medium, in different volumes (5 ml, 20 ml, 1 l) at different temperatures (25° C., 30° C. and 37° C.)

[0045] The culture media obtained were analysed by PAGE-SDS electrophoresis.

Results

[0046] All the proteins tested are expressed at a high level at 37° C. in the absence of inducer. As such, it can be observed on the PAGE-SDS electrophoreses that the heterologous protein is the protein most strongly expressed by the bacterium.

[0047] An accumulation of heterologous proteins is observed for at least 20 hours.

[0048] The production of heterologous proteins is not dependent on the volume of culture medium used.

[0049] These results are obtained at all temperatures compatible with bacterial growth.

[0050] The method according to the invention was implemented, with the same success, with heterologous proteins from mammals (particularly humans), bacteria and plants. Of these, mention can particularly be made of GSTs from humans (GSTA1 and GSTP1), drosophila (GSTD2 and GSTD7), a human lipocalin (LCN1), and human Hsp110.

[0051] The method according to the invention also functions regardless of the location of the protein: membranous, extracellular or cytoplasmic.

[0052] The method according to the invention is not dependent on the structure of the plasmid used and was successfully implemented with the plasmids pD431-SR, pD451-SR, pD434-SR, pD434-WR, pD454-WR, pJ431:2047, pJ414:2047 (DNA2.0 Menlo Park, USA).

Effects of Glucose and Lactose on the expression of the Proteins of Interest

Materials and Methods

[0053] The converted bacteria for expressing the recombinant proteins cited above were cultured in LB medium supplemented with 0.05% glucose or 0.2% lactose.

[0054] The bacterial growth was monitored by measuring the optical density at 600 nm and the quantity of recombinant protein produced was measured by PAGE-SDS chromatography of the culture supernatant.

Results

[0055] In the absence of glucose or lactose, the expression of the proteins of interest appears at an optical density of 1.

[0056] Adding 0.05% glucose in the medium delays the expression of the protein of interest to an optical density at 600 nm of 1.5.

[0057] Adding 0.02% lactose makes it possible to accelerate the production of the protein of interest to an optical density of 0.7.