Polypeptide having protease activity and methods for increasing its activity thereof

Abstract

The present invention relates to a polypeptide having protease activity comprising a zinc finger protease domain, a helix-turn-helix domain and a GAF domain. The core protein sequence of the protease is shown as SEQ ID NO: 1. The invention also relates to optimized reaction conditions for the protease and methods of increasing the protease activity.

Claims

1. A method for producing a composition comprising a polypeptide with increased protease activity where the polypeptide is isolated from Deinococcus radiodurans, comprising the following steps of: Step 1, preparing a proteolytic reaction buffer of 150 mM NaCl, 20 mM Tris-HCl PH 8.0, 1.0 mM DTT, 2.0 mM MnCl.sub.2; Step 2, adding a promoter comprising the DNA damage response gene containing the predicted radiation and desiccation resistance motif (RDRM) and a substrate consisting of transcription factor DdrO (Gene ID: 1798752; NP 296294.11) from Deinococcus radiodurans (ATCC No. 139391) to the proteolytic reaction buffer and incubate for a period of time to form a substrate buffer; and Step 3, adding the polypeptide to the substrate buffer and maintain a temperature in a range of 35-40 C., Wherein the polypeptide comprises a zinc peptidase-like domain containing polypeptide fragment of PR 1 (GI: 158052041) consisting of SEQ ID No: 1.

2. The method of claim 1, wherein the specific cleavage recognition sequence of said polypeptide is SEQ ID No: 2 (ELXGXR, where X is any kind of essential amino acids), and the cleavage site is between the second and the third amino acid residue.

3. The method of claim 1, wherein the specific cleavage recognition sequence is one of SEQ ID No: 3-8.

4. The method of claim 1, wherein gene promoter regions containing the RDRM site include dr0070, dr0099, dra0151, dr0219, dr0326, dra0346, dr0423, dr0596, dr0906, dr1039, dr1143, dr1289, dr1696, dr1771, dr1775, dr1913, dr1921, dr2256, dr2275, dr2336, and dr2574.

5. The method of claim 1, wherein the binding reaction between DdrO and the promoter regions containing the RDRM site is carried out in the buffer containing 150 mM NaCl, 20 mM Tris-HCl 8.0, 5 mM MgCl2 at 30 C.

6. The method of claim 1, wherein the minimum sequence for DdrO to bind to the promoter regions of the DNA damage response and repair gene is the RDRM site.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: PprI cleavage activity in vitro detected by SDS-PAGE: Lane 1 shows purified DdrO protein; Lane 2 shows purified PprI protein; Lane 3 shows proteolytic reaction of PprI; Lane 4 shows pre-stained protein marker (Fermentas, SM0671).

(2) FIG. 2: PprI cleavage activity detected by Western blotting: Lane 1 shows purified DdrO protein; Lane 2 shows proteolytic reaction between PprI and DdrO where PprI cleaves DdrO into two fragments.

(3) FIG. 3: C-terminal sequencing of the bigger cleaved fragment of DdrO: peak A shows the molecular weight of the smaller fragment; peak B shows the molecular weight of the bigger fragment; peak C shows the molecular weight of the complete substrate DdrO.

(4) FIG. 4: Alanine scan at the DdrO cleavage site to assay the sequence specificity; WT indicates the wildtype DdrO; Arg-109 and Lys-111 are mutated to Glu.

(5) FIG. 5: the cleavage model of PprI protease: amino acid residues in bold are essential for recognition and cleavage of the protease. X indicates the variable amino acid residue; the arrow points to the cleavage site.

(6) FIG. 6: metal ion scans for the PprI protease activity detected by SDS-PAGE: from left to right: without additive, EDTA, CaCl.sub.2, FeCl.sub.2, MgCl.sub.2, NiSO.sub.4, CuCl.sub.2, MnCl.sub.2, ZnCl.sub.2; the concentration of all the metal ions is 1 mM.

(7) FIG. 7: DdrO binding to the promoter regions containing RDRM in vitro: CK shows a negative control without adding DdrO; the other lanes show the binding of DdrO to the promoter regions of the DDR genes containing RDRM site.

(8) FIG. 8: shows the minimum DdrO binding sequence: seven promoters containing RDRM site were selected; PrecA (i.e. Pdr2340, etc.) shows the complete promoter with RDRM site, PrecA (i.e. Pdr2340, etc.) is the truncated promoter without the RDRM site. The promoters containing the RDRM site can be bound by DdrO, the promoters do not contain the RDRM site cannot be bound. The final concentration of DdrO is 0.8-2.4 M.

(9) FIG. 9: DdrO binds to the promoter regions of DDR genes in vivo: QRT-PCR was performed using the immunoprecipitated DNA. DNA fragments cross-linked to DdrO were enriched by rabbit anti-DdrO antibody. Nonspecific normal antibody of rabbit in ChIP assay was applied as a blank control. dr0089, a house-keeping gene, was used as a normalization factor.

(10) FIG. 10: RNA transcription of the wildtype strain after exposure to gamma radiation. Three time points were chosen: before radiation, recovery for 35 minutes and recovery for 90 minutes after the radiation.

(11) FIG. 11: RNA transcription of the pprI-knockout strain after exposure to gamma radiation. Three time points were chosen: before radiation, recovery for 35 minutes and recovery for 90 minutes after the radiation.

DETAILED DESCRIPTION OF THE INVENTION

(12) The present invention is further illustrated with the following specific examples, but the present invention includes but is not limited to the following steps and contents.

(13) The strains used in the invention are Deinococcus radiodurans (ATCC No. 13939), Escherichia coli expression strains BL21 (DE3) Chemically Competent Cell (Genetype: F-ompT hsdSB(rB-mB) gal dcm(DE3), Escherichia coli cloning strains Trans5 Chemically Competent Cell (Genetype: F-80 lac ZM15 (lacZY A-arg F) U169 endA1 recA1 hsdR17(rk,mk+) supE44-thi-1 gyrA96 relA1 phoA).

Embodiment 1

(14) (1). The Proteolytic Activity and Recognition Sequence Specificity of PprI Protease

(15) The protease activity of PprI was performed in vitro by incubating its substrate DdrO with PprI for 40 minutes. The final reaction buffer was 150 mM NaCl, 20 mM Tris-HCl pH 8.0, 1 mM DTT, and 2.0 mM MnCl.sub.2. The reaction product was detected by SDS-PAGE. DdrO was cleaved by PprI into two fragments. Moreover, through point mutation of the amino acid residues around the DdrO cleavage site, the specific recognition sequences of PprI protease digestion were detected, and they are:

(16) TABLE-US-00003 SEQIDNO:3: ELRGKR SEQIDNO:4: ELRGAR SEQIDNO:5: ELRGER SEQIDNO:6: ELAGKR SEQIDNO:7: ELAGAR SEQIDNO:8: ELAGER

(17) In addition, the cleavage site was detected to locate between the second and the third amino acid residues by C-terminal sequencing of the larger cleaved fragment (FIGS. 1-5).

(18) (2). The Optimum Temperature Range and Temperature Resistance of PprI Protease

(19) The optimum temperature range of PprI cleavage activities were between 35 C. and 40 C. When the temperature was between 50 C. and 55 C., the protease activity still existed, but decreased to one third of the optimum activity. The activity was further decreased at 65 C.

(20) (3). Increasing the PprI Cleavage Activity by Optimizing the Manganese Ion Concentration

(21) PprI protease activity was increased by the presence of Me and the optimum final concentration of Mn.sup.2+ was 2 mM (FIG. 6).

(22) (4). Increasing the PprI Cleavage Activity by Optimizing the DdrO Binding Activities to the Promoter Regions Containing RDRM Site In Vitro

(23) The promoter region of dr2340 was added to the binding buffer (200 mM NaCl, 50 mM Tris-HCl 8.0, 10 mM MgCl.sub.2) without DdrO for 40 minutes. The product was detected by 12% TB-PAGE. The experiment showed that the DNA band did not shift when DdrO protein was not added (FIG. 7).

(24) (5). The RDRM Site of the Gene Promoter Regions is Essential for Increasing the PprI Cleavage Activity by Optimizing the DdrO Binding Activities In Vitro

(25) The promoter regions of dr0326, dra0346 and dr2574 were reacted with the DdrO in the binding buffer (200 mM NaCl, 50 mM Tris-HCl PH 8.0, 10 mM MgCl.sub.2) for 40 minutes. The EMSA experiments showed that all the promoter regions could bind to DdrO (FIG. 8).

(26) (6). DdrO Binds to the Promoter Regions Containing RDRM Site In Vivo

(27) Chromatin-immunoprecipitation assay was performed. DNA fragments cross-linked to DdrO were enriched by rabbit anti-DdrO antibody. QRT-PCR analysis showed that transcription of dr0070 and dr0099 in wildtype strain R1 was up-regulated significantly after exposure to radiation. Nonspecific normal antibody of rabbit in ChIP assay was applied as a blank control. Dr0089 was used as a normalization factor in qRT-PCR (FIG. 9).

(28) (7). The Transcription Level of DDR Genes in Wildtype Strain R1 and pprI-Knockout Strain YR1 Before Exposure to Radiation

(29) Before exposure to radiation, wildtype strain R1 and pprI-knockout strain YR1 were collected, followed by RNA extraction, reverse transcription, and qRT-PCR. The results showed that transcription levels of dr2340, dr2574, dr0070, dra0346, dr0423, dr0090 and dr1289 were unchanged in the mutant YR1 relative to the wildtype R1 before exposure to radiation. dr0089 was used as a normalization factor (FIGS. 10-11).

Embodiment 2

(30) The protease activity of PprI was performed in vitro by incubating its substrate DdrO with PprI for 40 minutes. The final reaction buffer was 100 mM NaCl, 30 mM Tris-HCl pH 8.0, 1 mM DTT, and 3.0 mM MnCl.sub.2. The reaction product was detected by SDS-PAGE, and DdrO was cleaved by PprI into two fragments. Moreover, through point mutation of the amino acid residues around the DdrO cleavage site, the specific recognition sequences of PprI protease digestion were detected, and they are:

(31) TABLE-US-00004 SEQIDNO:3: ELRGKR SEQIDNO:4: ELRGAR SEQIDNO:6: ELAGKR SEQIDNO:7: ELAGAR

(32) In addition, the cleavage site was detected to locate between the second and the third amino acid residues by C-terminal sequencing of the larger cleaved fragment (FIGS. 1-5).

(33) (2). The Optimum Temperature Range and Temperature Resistance of PprI Protease

(34) The optimum temperature range of PprI cleavage activities were between 35 C. and 40 C. The protease activity remained the highest during this temperature range and was consistent. When the temperature was 4 C., the protease activity still existed, but was weaker. When the temperature was 65 C., the protease activity was also weaker.

(35) (3). Increasing the PprI Cleavage Activity by Optimizing the Manganese Ion Concentration

(36) PprI protease activity requires the presence of Mn.sup.2+ and was increased to the optimum level when the final concentration of Mn.sup.2+ was 2 mM. When the final concentration of the other metal ions (such as Ni.sup.2+, Zn.sup.2+) was higher than 0.25 mM, the cleavage activity was inhibited (FIG. 6).

(37) (4). Increasing the PprI Cleavage Activity by Optimizing the DdrO Binding Activities to the Promoter Regions Containing RDRM Site In Vitro

(38) DdrO and the promoter regions of dr0070, dr0099, dra0151, dr0219, dr0326, dra0346, dr0423 dr0596, dr0906 and dr1039, respectively, was added to the binding buffer (200 mM NaCl, 50 mM Tris-HCl 8.0, 5 mM MgCl.sub.2) for 40 minutes. The products were detected by 12% TB-PAGE, the experiment showed that the DNA bands shifted when each of the promoters was added to the DdrO protein (FIG. 7).

(39) (5). The RDRM Site of the Gene Promoter Regions is Essential for Increasing the PprI Cleavage Activity by Optimizing the DdrO Binding Activities In Vitro

(40) The promoter regions that do not contain the RDRM site, Pdr0070-, Pdr0099-, Pdr2338- and Pdr0423-, were reacted with DdrO in the binding buffer (200 mM NaCl, 50 mM Tris-HCl PH 8.0, 5 mM MgCl.sub.2) for 40 minutes. The products were detected by 12% TB-PAGE. The experiment showed that the DNA bands did not shift when each of the promoters was added to the DdrO protein (FIG. 8).

(41) (6). DdrO Binds to the Promoter Regions Containing RDRM Site In Vivo

(42) Chromatin-immunoprecipitation assay was performed. DNA fragments cross-linked to DdrO were enriched by rabbit anti-DdrO antibody. The transcriptions of dr0326 and dra0346 were detected by qRT-PCR. The results showed that the quantity of selected promoters enriched by specific anti-DdrO antibody were 3 to 6 fold higher than that enriched by non-specific antibody (FIG. 9).

(43) (7). The Transcription Level of DDR Genes are Up-Regulated in Wild Type Strain R1 Relative to the PprI-Knockout Strain YR1 after Exposure to Radiation

(44) After exposure to 2 KGy gamma radiation, wild-type strain R1 and pprI-knockout strain YR1 were recovered in the fresh media for 35 minutes and collected, followed by RNA extraction, reverse transcription, and qRT-PCR. The results showed that the transcription levels of dr2340, dr2574, dr0070, dra0346, dr0423, dr0090 and dr1289 were up-regulated after exposure to gamma radiation in wild-type R1, while the transcription level was unchanged in the pprI mutant YR1. The house-keeping gene, dr0089, was used as a normalization factor (FIGS. 10-11).

Embodiment 3

(45) The protease activity of PprI was performed in vitro by incubating its substrate DdrO with PprI for 40 minutes. The final reaction buffer was 150 mM NaCl, 20 mM Tris-HCl pH 8.0, 1 mM DTT, and 5.0 mM MnCl.sub.2. The reaction product was detected by SDS-PAGE, and DdrO was cleaved by PprI into two fragments. Moreover, through point mutation of the amino acid residues around the DdrO cleavage site, the specific recognition sequences of PprI protease digestion were detected, and they are:

(46) TABLE-US-00005 SEQIDNO:3: ELRGKR SEQIDNO:4: ELRGAR SEQIDNO:5: ELRGER SEQIDNO:6: ELAGKR SEQIDNO:7: ELAGAR SEQIDNO:8: ELAGER

(47) In addition, the cleavage site was detected to locate between the second and the third amino acid residues by C-terminal sequencing of the larger cleaved fragment (FIGS. 1-5).

(48) (2). The Optimum Temperature Range and Temperature Resistance of PprI Protease

(49) The optimum temperature range of PprI cleavage activity was between 35 C. and 40 C. When the temperature was within the range, the protease remained the highest activity. The activity was relatively weaker at 4 C. When the temperature was between 50 C. and 55 C., the protease activity still existed, but decreased to one third of the optimum activity.

(50) (3). Increasing the PprI Cleavage Activity by Optimizing the Manganese Ion Concentration

(51) PprI protease activity requires the presence of Mn.sup.2+. When the final optimum concentration was 5 mM, the activity was still existed. When the final concentration of the other metal ions (such as Fe.sup.2+, Cu.sup.2+) was higher than 0.25 mM, the cleavage activity was inhibited (FIG. 6).

(52) (4). Increasing the PprI Cleavage Activity by Optimizing the DdrO Binding Activities to the Promoter Regions Containing RDRM Site In Vitro

(53) The promoter region of dr2340 was added to the binding buffer (200 mM NaCl, 50 mM Tris-HCl 8.0, 10 mM MgCl.sub.2) without DdrO for 40 minutes. The product was detected by 12% TB-PAGE. The experiment showed that the DNA band did not shift when DdrO protein was not added (FIG. 7).

(54) (5). The RDRM Site of the Gene Promoter Regions is Essential for Increasing the PprI Cleavage Activity by Optimizing the DdrO Binding Activities In Vitro

(55) The promoter regions of dr0326, dra0346 and dr2574 were reacted with DdrO in the binding buffer (200 mM NaCl, 50 mM Tris-HCl PH 8.0, 10 mM MgCl.sub.2) for 40 minutes, and then detected by 12% TB-PAGE. The EMSA experiments showed that all the promoter regions could bind to DdrO (FIG. 8).

(56) (6). DdrO Binds to the Promoter Regions with RDRM Site In Vivo

(57) Chromatin-immunoprecipitation assay was performed. DNA fragments cross-linked to DdrO were enriched by rabbit anti-DdrO antibody. The transcription of negative control, dr0089 was detected by qRT-PCR. The result showed that the quantity of dr0089 promoter enriched by specific anti-DdrO antibody was consistent with that enriched by non-specific antibody (FIG. 9).

(58) (7). The Transcription Levels of DDR Genes in Wild Type Strain R1 and PprI-Knockout Strain YR1 During the Post-Recovery Period

(59) After exposure to 2 KGy gamma radiation, wildtype strain R1 and pprI-knockout strain YR1 were recovered in the fresh media for 90 minutes and collected, followed by RNA extraction, reverse transcription, and qRT-PCR. The results showed that transcription levels of dr2340, dr2574, dr0070, dra0346, dr0423, dr0090 and dr1289 were unchanged in both the wild-type R1 and the pprI mutant YR1 during the post-recovery period. The house-keeping gene, dr0089, was used as a normalization factor (FIGS. 10-11).

(60) Strain used in the above embodiments is Deinococcus radiodurans (ATCC No. 13939). Furthermore, according to the teachings and enlightenment of the present invention, any synthetic or other natural protease and derivatives, such as PprI homologous sequence, similar structure and function, is also within the protection scope of the present invention.

(61) Finally, it should be declared that the above examples are merely used to help those skilled in the art to understand the present invention, rather than to limit the protection scope of the present invention, and any relevant technical solutions obtainable by those skilled in the art according to general technical knowledge and common knowledge fall within the protection scope of the present invention.