Patent Application
20120196328
The entirety of the Sequence Listing submitted at the same time of the filing of the instant application is incorporated by reference herein.
The present invention relates to the technique of recombinant fusion protein, specifically relates to a protein mixture. In addition, the invention relates to a preparation method of the protein mixture.
In 2006, Japanese Yamanaka laboratory obtained a kind of pluripotent stem cell successfully, which was very similar to mouse embryonic stem cells in characteristics, by transducing four transcription factors (KLF4, c-Myc, SOX2, OCT-4) into mouse embryonic stem cells and adult fibroblast through retrovirus. Soon, using the same method, human fibroblast was transduced to induce human pluripotent stem cells successfully. Afterwards, a variety of IPS cells induced from patient cells with hereditary disease were obtained successfully. IPS cells are similar to embryonic stem cells in characteristics. The embryonic stem cells can differentiate into all types of somatic cells, which are able to be used to repair the tissue injury from diseases or hurt. So the embryonic stem cells have a very extensive application prospect in the field of regenerative medicine. However, application of embryonic stem cells in medicine has two important obstacles: one is immunologic rejection after transplantation, the other is the ethical consideration of using human embryo. Even if the embryonic stem cells were obtained through somatic cell nuclear transfer, there are still problems of ethics.
Nevertheless, the human IPS cells can be obtained from patient cells, which does not have the problem of immunologic rejection. And since no destroying human embryo or using human ootids, the ethical problem of using embryonic stem cells does not exist. These advantages above enable the IPS technology to have a better application prospect in regenerative medicine.
Initially, inducing Pluripotent Stem cells needed to use replication-deficient retrovirus or Lentiviral Vector, which could transduce the reprogramming factors into cells. These viral vectors would integrate into genome of the host cell. Although these exogenous genes are silent in IPS cells at most cases, once being reactivated, they will induce tumor. Leaked expression of these genes also possibly enables IPS cells to differentiate and ripe incompletely, resulting in the increasing risk of forming immaturity teratoma. Viral integration also possibly activates or terminates expression of endogenous genes. In the history of gene therapy, using the technology of retrovirus integration resulted in leukemia because of activating oncogenes. Many laboratories tried to use the technology of non-viral integration to induce Pluripotent Stem cells. Adenovirus and/or plasmid were used as vectors to introduce reprogramming factors into cells, thus obtained IPS cells successfully. But the rate of obtaining IPS cells was very low. OriP/EBNA-1 plasmid episome was used as vector to induce IPS cells. Some laboratories used Cre/loxp, transposon/transposase, to remove the exogenous gene being integrated into genome after obtaining IPS cells. Another method to avoid integration of exogenous genes was to replace reprogramming factors by chemicals. So far, from the results published, no completely alternative reprogramming transcription chemicals or combination has been found, only one or two reprogramming factors could be replaced. Even though no foreign gene was integrated into IPS cells, the method mentioned above could not completely avoid genomic change, for instance, the method of plasmid transformation would bring genomic integration with low chance, while chemicals could lead to gene mutation.
Initially, protein transduction was originated from the research of HIV TAT protein. People found that the entire HIV TAT protein could enter cells to activate transduction of viral gene. Further studies showed that a region (TAT PTD) of HIV TAT protein was responsible for the function of entering cells. Coupling or fusion of TAT PTD and macromolecules was found useful for the macromolecules to enter cell. Study showed arginine with positive charges was necessary for TAT PTD to enter cytoplasm through cell membrane. Any mutation of an arginine would lead to loss of the transduction function. Based on this, polyarginine was found to possess the function of transduction similarly. The technology of protein transduction by TAT PTD or the other protein transducing peptide has a great potential for macromolecular pharmaceuticals to enter cells and exert their function.
Small ubiquitin-like modifier (SUMO) could covalently modify protein. SUMO modification can regulate various cell process including nuclear transfer, signal transduction and stability of protein. Ulp1, a SUMO protease, could specifically recognize the tertiary structure of SUMO, and cleave at the joint of SUMO and its modified protein. When SUMO fusing with other protein (equivalently SUMO modifies N-terminal amino of target protein), Ulp1 could specifically remove SUMO, and release the target protein completely.
The reprogramming factors (KLF4, c-Myc, SOX2, OCT-4) were introduced into cells by the technology of protein transduction, which can avoid the security problems mentioned above. A laboratory of Scripps institute successfully obtained mouse IPS cells by a recombinant protein of four reprogramming factors expressed in E. coli, with the addition of a kind of chemical (HDAC inhibitor). The recombinant reprogramming factors used in the laboratory were OCT-4, KLF4, Sox2, c-Myc. The four reprogramming factors were introduced into cells by polyarginine with C-terminal fusion. The technology can completely overcome all disadvantages when the reprogramming factors were introduced into cells by using DNA, thus enables the possible application of IPS cells in Regenerative Medicine to take a significant step forward. However, the protein transduction peptide—polyarginine fused the reprogramming factors directly. Since the transduction peptide carried strong positive charges, which possibly nonspecifically bind with genomic DNA carrying negative charges, thus the transcription factor fused with transduction peptide could nonspecifically change gene expression. The nonspecific change of gene expression could lead to low rate of inducing Pluripotent Stem Cells or some permanent change of gene expression, thus influenced subsequent differentiation and maturity of the IPS cells. Hence, obtaining a preparation, which could overcome the disadvantages above in inducing Pluripotent Stem Cells, had a great significance for human IPS cells to use practically in Regenerative Medicine.
The technical problem to be solved in this invention is to provide a protein mixture with potential value of medical application. The protein mixture can greatly decrease the possibility of nonspecifically combining with genomic DNA, which has activity of transcription activation after being transduced into cells. It can be used to induce Pluripotent Stem Cells. Moreover, the invention provides a method for preparing the protein mixture.
In order to solve the technical problems mentioned above, the invention provides the following technical solutions:
The invention provides a protein mixture, which consists of a fusion protein of C-myc, SOX2, KLF4 and OCT-4. The applied concentration for each fusion protein is ing/ml-1 mg/m. The fusion proteins comprise the structure: PTD-SUMO-Protein, wherein, PTD is a Protein Transduction Domain, standing for HIV-TAT, HSV-VP22, AntP or poly-arginine; SUMO is a small ubiquitin-like modifier, standing for yeast SMT3p or its homologs in other species, which is the recognizing region after the fusion protein enters cells and is cleaved; Protein is C-myc, SOX2, KLF4 or OCT-4.
Protein mentioned above is TAT PTD region of HIV TAT protein, or other amino acid sequences with protein transduction function. The Protein Transduction Domain (PTD) enables the protein mixture to enter human cells.
Small ubiquitin-like modifier (SUMO) mentioned above is yeast SMT3p or other amino acid sequences, whose tertiary structure can be recognized and cleaved by SUMO protease. The small ubiquitin-like modifier (SUMO) enables the protein transduction peptide to be removed from the fusion protein.
Preferably, NES is inserted between the mentioned PTD and SUMO. The mentioned fusion protein has the structure: PTD-NES-SUMO-Protein, NES is an optional Nuclear Export Sequence, which decides cytoplasmic localization of the fusion protein.
The mentioned optional Nuclear Export Sequence is human IkBa Nuclear Export Sequence, or the amino acid sequences with function of Nuclear Export Sequence.
The mentioned PTD-NES-SUMO has DNA sequence as shown in SEQ ID NO.1, and has amino acid sequence as shown in SEQ ID NO.2.
The mentioned PTD-NES-SUMO-SOX2 has DNA coding sequence as shown in SEQ ID NO.3, PTD-NES-SUMO-OCT-4 has DNA coding sequence as shown in SEQ ID NO.4, PTD-NES-SUMO-KLF4 has DNA coding sequence as shown in SEQ ID NO.5, PTD-NES-SUMO-C-myc has DNA coding sequence as showed in SEQ ID NO.6.
Preferably, 6 histidines are inserted between PTD and NES for purification.
In addition, the invention provides a preparation method of the protein mixture, including the following steps:
(1) Constructing the expression plasmid of PTD-NES-SUMO-Protein: First, respectively synthesize 8 oligo primers which are 75 bp in length and mutually overlap in 20 bp. After three times of overlapping PCR, PTD-NES-SUMO is synthesized. Then, human OCT-4, SOX2, KLF4, C-myc cDNA are obtained from amplification of total RNA of human embryonic stem cells, whose 3′-end has an XhoI site, which were assembled by PCR with the synthesized TAT-NES-SUMO sequence. The product is cloned in NdeI/XhoI site of pET-24a (+) vector.
(2) Screening the expression strain: PTD-NES-SUMO-Protein expression plasmid obtained from the step 1 is transformed into host strain BL21, to culture and screen high-expression clone with mini-scale.
(3) Large-scale expression of the fusion protein: the expression strain is inoculated in flasks, and cultured until OD 0.6, then IPTG is added to induce for 3 hours.
(4) Separation and purification of the fusion protein: the fusion protein mentioned above is separated and purified by hydrophobic chromatography and ion exchange chromatography.
The protein mixture prepared by the invention, can fundamentally solve the present technology deficiencies (the nonspecific change of gene expression could lead to low rate of inducing Pluripotent Stem Cells or some permanent change of gene expression, thus influenced subsequent differentiation and maturity of the IPS cells). The fusion of PTD enables the fusion protein to enter cells. SUMO in the fusion protein enables protein transduction peptide to be cleaved from the fusion protein, to remove the fused PTD, NES, SUMO. If the mixture is used to induce Pluripotent Stem Cells, it can minimize the adverse effects brought by adding inducer for obtaining IPS cells during the course of inducing Pluripotent Stem cells, which adapts the IPS cells for application in regeneration medicine and other related fields.
The invention will be explained below in closer detail by reference to the schematic drawings and examples. Although the present invention has been described in detail, it should be understood that various changes, substitutions and alternations can be made hereto without departing from the spirit and scope of the invention. The experimental methods without detailed conditions mentioned should be conducted following the routine conditions, for example: conditions listed in the laboratory handbook: Sambrook et al., molecular clone: (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the instructions advised by manufacturers.
The codons of HIV TAT PTD, yeast SMT3p (SUMO) and human IkBa Nuclea Export Sequence (NES) are optimized according to the amino acid sequence listed in prior literatures, for the purpose of high-level expression in E. coli. TAT-NES-SMT3p are arranged in sequence, and 6 histidines are inserted between TAT and NES for purification. Encoding sequences of this part of fusion protein are synthesized by PCR assembling based on oligonucleotide, and a NdeI site is at its 5′-end. The methods are as follow: synthesize respectively 6 oligo primers which are 75 bp in length and mutually overlap in 20 bp. After three times of overlapping PCR (as shown in
Use Invitrogen Trizol Reagent to extract the total RNA from 5×106 human embryonic stem cells. According to the instruction (Invitrogen) of Superscript III reverse transcription PCR kit, RNA is reverse transcribed into cDNA using random primer. cDNA of OCT-4, SOX2, KLF4, C-myc are obtained by amplification using the following primers:
In these primers, all their 5′-end contain 20 bases being the same as the 3′-end of TAT-NES-SMT3p fragment, which is convenient for assembling.
Each cDNA is assembled with the synthetic TAT-NES-SUMO sequence by PCR (the reaction conditions are same as those for assembling TAT-NES-SUMO, using primer a and 3′ primer of each cDNA). The product is cloned into vector pET-24a(+) (Novagen) at NdeI/XhoI site.
TAT-NES-SUMO has DNA coding sequence shown in SEQ ID NO.1.
TAT-NES-SUMO has amino acid sequence shown in SEQ ID NO.2.
TAT-NES-SUMO-50×2 has DNA coding sequence shown in SEQ ID NO.3.
TAT-NES-SUMO-OCT-4 has DNA coding sequence shown in SEQ ID NO.4,
TAT-NES-SUMO-KLF4 has DNA coding sequence shown in SEQ ID NO.5,
TAT-NES-SUMO-C-myc has DNA coding sequence shown in SEQ ID NO.6
The fusion expression plasmid is transformed into host strain BL21 (DE3). Screen high-expression clone by mini-scale culture. In 3 ml E. coli of OD 0.6, 0.1 mM IPTG is added to induce expression for 3 hours. The thalli are collected and sampling buffer is added to boil for 5 minutes. Then SDS polyacrylamide gel electrophoresis and Coomassie Brilliant Blue Stain are used, select high-expression clone as seed for large-scale expression. By further analysis on the high-expression strain, most of the expressed fusion protein is found in inclusion bodies.
The expression strain is inoculated in 10 L LB media, cultured at 37° C. till OD600 0.6, then 0.1M IPTG is added to induce for 3 hours. Then the concentration reaches OD600 1.0.
The cultures above are centrifuged to remove media, and get about 27.4 g thalli. 300 ml lysis buffer (50 mM PH 8.0 Tris-Cl, 500 mM NaCl) is added to resuspend the thalli. Then the thalli are split using ultrasonic at 4° C., then centrifuged at 6000 rpm in low temperature, to remove supernatant. 300 ml lysis buffer is used to wash the precipitation, centrifuge; remove supernatant. Dissolve the inclusion bodies with solubilized buffer (50 mM PH 8.0 Tris-Cl, 500 mM NaCl, 8M Urea). After the inclusion bodies are dissolved, affinity chromatography is performed for purification through IMAC. After sample-loading, rinse solution (8M Urea, 500 mM NaCl, 50 mM Tris-HCl pH8.0, 20 mM Imidazole) is used to wash the components combined nonspecifically. Then (8M Urea PH 8.0 50 mM Tris-Hcl, 500 mM NaCl 250 mM Imidazole) is used to elute, meanwhile detecting 280 nM ultraviolet absorbance. The protein peak is collected. 80 ml eluent is obtained in all.
Hydrophobic chromatography. Solid NaCl is added in the elution buffer above to 2M, dissolved fully, centrifuges at 1000 RPM for 15 minutes and discards the pellet. The supernatant is loaded onto Phenyl Sepharose FF column equilibrated fully by equilibration buffer (2M NaCl, 50 mM Tris-HCl pH8.0). After loading, 3 column volumes of equilibration buffer is used to rinse, the target peak is eluted by elution buffer (50 mM Tris-HCl pH8.0).
The target protein is regulated to pH6.0 with 1M acetic acid, and diluted threefold with pyrogen-free water, which is loaded onto SP-HP column equilibrated fully with equilibration buffer (10 mM NaAc—HAc,pH6.0). Eluted with gradually-increasing NaCl concentration, the target protein collected is purified fusion protein. After filtration sterilization through 0.22 nm microfiltration membrane, it goes to functional detection.
Hela cells are cultured in high-glucose DMEM (10% fetal calf serum). When the cells overspread 30%, TAT-NES-SUMO-reprogramming factors are added (concentration of every protein is 5 ug/ml). After 12 hours, change the culture medium and continue to culture for 12 h, 24 h, 72 h. Wash cells with cold PBS two times. Split the cells with lysis buffer (PH 7.5 20 mM Tris-Cl, 200 mM NaCl, 1% NP-40, 1 mM PMSF). Cell lysis buffer with 40 ug total protein is added 5× sample-buffer and goes to SDS polyacrylamide gel electrophoresis, then it is transferred to PVDF membrane. Anti-OCT4, SOX2, c-Myc, (cell signaling) KLF4 (santa Cruz) are used in Western blot experiment.
Results indicates (showed in
The experimental course is same as that in example 2. Results indicates (shown in
OCT4 reporter gene plasmid: 8 tandem OCT4 binding sites (ATGCAAAT) Primer A (SEQ ID NO.21) primer B (SEQ ID NO.22) anneal, and are inserted in KpnI/BglII site of PGL3-promoter luciferase plasmid.
KLF4 reporter gene plasmid: 8 tandem KLF4 binding sites (AGGGTGC). Primer A (SEQ ID NO.23) primer B (SEQ ID NO.24) anneal, and are inserted in KpnI/BglII site of PGL3-promoter luciferase plasmid.
Sox2 reporter gene plasmid: Hesx1 gene. In the upstream 570-bp of Hesx1 translation initiation sites, PCR fragment is inserted into KpnI/SmaI site of pGL3-basic vector (Promega) by KpnI restriction digestion. Primer 5′-CGAGGTACCGAGTTCTCTGTTCTATAAAC-3′(SEQ ID NO.25) and 5′-CGACCCGGGCCTCTCGTGGTCTGCACAGA-3′ (SEQ ID NO.26).
C-myc reporter gene plasmid: Primer A (5′-CCGGTACCGG GTTGTGGCAG CCAGTCACGT GCCCGCCGCG TAGCCACACC TCTGCTCCTC AGAGCAATGT CAAGCGGTCA CGTGTGATAG CAACAGATCA CGTGGCTGCC ATCGCCCCTC-3′) (SEQ ID NO.27) and primer B (5′-ATGAATTCCG GACGTTCTGG GCACGTGACC GCCACCCATG CGCTGAGGGG CGGACAGGAG GTGCTTCGAC TGGGAGGAGG GCGAAGAGTG TAAGGGGGCG GAGGGGCGAT GGCAGCC-3′) (SEQ ID NO.28) anneal, and are inserted into KpnI/SmaI site of PGL3-promoter luciferase plasmid by KpnI restriction digestion.
When Hela cells are cultured to overspread 50%, in the 12-well cell plate, fugene6 (from Roche) is used to transfect transcription reporter gene (firefly luciferase) and reference reporter plasmid pRL-TK-luc (promega, Renilla luciferase) of the reprogramming factors. After transfecting 6 hours, change media, add 5 ug/ml fusion protein. Then continue to culture till 12 h, 24 h, 48 h, 72 h. Wash cells with cold PBS, split cells with reporter lysis buffer (from promega). Luciferase activity of the cell lysis buffer is detected with dual-luciferase kit from Promega.
Results indicated (showed in
| Number | Date | Country | Kind |
|---|---|---|---|
| 200910057744.6 | Aug 2009 | CN | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/CN10/01223 | 8/12/2010 | WO | 00 | 2/10/2012 |
