Công nghệ chuyển protein và sự tổng hợp một peptid Runx 3 có thể di chuyển trực

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Công nghệ chuyển protein và sự tổng hợp một peptid Runx 3 có thể di chuyển trực tiếp vào tế bào Pham Thanh Van, Suk-Chul Bae Viện nghiên cứu Ung thư, trường Đại học Quốc Gia Chung Buk, Hàn Quốc. Sự vận chuyển những phân tử lớn như protein hay polipeptid vào tế bào động vật vốn gặp nhiều khó khăn do sự cản trở của màng tế bào. Tuy nhiên trong 10 năm trở lại đây, một phương pháp mới đã được đề xuất bởi nhóm nghiên cứu Steven Dowdy để giải quyết khó khăn trên, đó là công nghệ chuyển...

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Nội dung Text: Công nghệ chuyển protein và sự tổng hợp một peptid Runx 3 có thể di chuyển trực

TRƯỜNG …………………. KHOA………………………. ----- ----- Công nghệ chuyển protein và sự tổng hợp một peptid Runx 3 có thể di chuyển trực
Công nghệ chuyển protein và sự tổng hợp một peptid Runx 3 có thể di chuyển trực tiếp vào tế bào Pham Thanh Van, Suk-Chul Bae Viện nghiên cứu Ung thư, trường Đại học Quốc Gia Chung Buk, Hàn Quốc. Sự vận chuyển những phân tử lớn như protein hay polipeptid vào tế bào động vật vốn gặp nhiều khó khăn do sự cản trở của màng tế bào. Tuy nhiên trong 10 năm trở lại đây, một phương pháp mới đã được đề xuất bởi nhóm nghiên cứu Steven Dowdy để giải quyết khó khăn trên, đó là công nghệ chuyển protein (protein transduction technology). Công nghệ chuyển protein cho phép vận chuyển trực tiếp protein hay các cao phân tử vào trong tế bào bằng cách dung hợp phân tử cần nghiên cứu với một trình tự đặc biệt có khả năng vượt qua màng tế bào vào nội bào. Những trình tự này đã được phát hiện đầu tiên trên protein Tat của HIV 1, Antp của ruồi giấm và HSV VP22 của virus HSV, và được đặt tên là "vùng chuyển protein"
(protein transduction domains). Dựa trên các kết quả thực nghiệm, các nhà khoa học cho rằng nếu protein bị biến tính trước khi đưa vào môi trường nuôi cấy tế bào thì quá trình chuyển protein sẽ xảy ra nhanh hơn và hiệu quả lớn hơn rất nhiều so với việc vận chuyển protein tự nhiên. Sau khi đã vào tế bào, hệ thống chaperone trong tế bào sẽ đưa protein biến tính trở về dạng tự nhiên ban đầu và hoạt động bình thường. Tuy nhiên, vì cơ chế hoạt động của chaperone vẫn chưa được xác định rõ ràng, cách thức này có một trở ngại là với những protein chưa rõ chức năng, sẽ khó có thể kết luận được sự bất hoạt của nó trong tế bào là do nó không có được chức năng như ta phỏng đoán hay do chaperone của tế bào đã không hồi phục được protein từ dạng biến tính trở về cấu trúc tự nhiên ban đầu. Bài báo này muốn giới thiệu thử nghiệm đưa protein ở dạng tự nhiên, không bị biến tính vào một số dòng tế bào động vật, mặc dầu phương pháp này đã được đánh giá là sẽ vấp phải nhiều khó khăn. Đối tượng nghiên cứu là trình tự gồm
30 axit amin ở đầu cuối của protein Runx3 - một thành viên trong gia đình Runx đóng vai trò như những tác nhân điều hoà nghiêm ngặt liên quan tới sự hình thành mô mới và sự chết của tế bào. Bằng cách dung hợp đoạn peptid cần nghiên cứu với "vùng chuyển protein" Tat, chúng tôi đã đưa được protein vào 2 dòng tế bào nguyên bào sợi NIH/3T3 và nguyên bào cơ C2C12. Sự chuyển protein đạt hiệu quả cao nhất ở nồng độ protein tối thiểu là 400ug/ml. esearch, Chungbuk National University A. An introduction of Protein Transduction Technology. The delivery of large, hydrophilic molecules such as proteins and oligonucleotides to the cytoplasm and nucleus of cells is problematic due to their poor plasma membrane permeability. Manipulation of protein expression in mammalian cells has been achieved by microinjection, electroporation or transfection of expression vectors. While these approaches have been somewhat successful, the
classic manipulation methods are not easily regulated and can be laborious due to the limitations of each method. One approach to circumvent these problems is Protein Transduction Technology, a new method that allows the direct entry of protein into mammalian cells. Protein Transduction Technology. Protein transduction was first reported in 1988 by Green [1] and Frankel [2], who independently demonstrated that the full-length (86-amino-acid) TAT protein from the HIV-1 virus was able to enter cells when added to the surrounding media and afterwards transactivate the viral LTR promoter. Consequently, in 1991, Frankel suggested that TAT might prove a useful vehicle to deliver proteins or peptides into cells and later, in 1994, HRP and -galactosidase chemically crosslinked to a 36-amino-acid domain of TAT were shown to transduce into cells. Subsequent to the TAT discovery, other proteins processing the ability to transduce have been identified, including Drosophila Antennapedia
(Antp) homeotic transcription factor and the herpes- simplex-virus-1 DNA-binding protein VP22. In all of these above proteins, the activity of translocating across cellular membranes is confined to a short stretch of less than 20 amino acids. These sequences are called "protein transduction domains" (PTDs) [3] (Table.1). The minimal TAT transduction domain is the basic residues 47- 57, whereas residues 267-300 of VP22 and the third alpha helix (residues 43-58) of the ANTP homeodomain are required for transduction. Table 1. Amino acid sequence of characterized PTDs (S.Dowdy, 2000). HIV-1 TAT: transcription-activating factor involved in the replication of HIV-1; HSV VP22: herpes-simplex-virus-1 DNA-binding protein VP22; Antp:
Drosophila Antennapedia (Antp) homeotic transcription factor. Significant efforts have been taken to advance this phenomenon into a broadly applicable method that allows for the rapid introduction of full-length proteins into primary and transformed cells. The first convenient method to apply the protein delivery potential of Tat was developed by the group of Steven Dowdy [4]. The technology requires the synthesis of a fusion protein, linking the TAT transduction domain to the molecule of interest using a bacterial expression vector, followed by the purification of this fusion protein through a series of affinity and desalt columns. The purified fusion protein is made soluble in an aqueous buffer and can be directly added to mammalian cell culture medium. One of the main advantages of protein transduction is that, by varying the amount of protein added to the culture medium, researchers can control the final intracellular
protein concentration. In addition, the process is specific since only peptides fused with a PTD can cross the plasma membrane. Finally, whereas some mammalian cells are notoriously difficult to transfect, all mammalian cells tested to date are receptive to protein transduction. With the above advantages, protein transduction technology has the potential of becoming a useful and broadly applicable tool in biological research and especially, in molecular medicine. It is possible that protein delivery will have some application to current gene therapy protocols, in cases where the direct delivery of the gene product itself may be more beneficial than the delivery of the gene. In addition, the technology has potential drug delivery applications and may be applied in vaccine development. With the completion of the human genome project, protein transduction technology promises the ability of determining the function of newly identified proteins. B. Synthesis and transduction of native Runx3
terminal peptide into some mammalian cell lines. I. Introduction. Despite new possibilities promised by PTDs, the exact mechanism of these domains across membrane remains poorly understood. It has been reported that because of reduced structural constraints, high energetic, denatured proteins may transduce much more efficiently into cells than low energetic, correctly folded proteins [4,5]. Once inside the cells, denatured proteins may be correctly folded by chaperones such as HSP 90 [6]. Although this hypothesis seems experimentally true and as a matter of fact, most of studies up to now have prepared denatured proteins for their transduction assays, the mechanism of chaperone activity is yet unestablished and it is not guaranteed that chaperones operate appropriately every time. It leads to an obstacle that in experiments to identify the function of a protein, it may be difficult to confirm whether the protein does not have the function we assumed, or denatured protein has not been folded precisely.
Figure 1. Internalization model of denatured protein into cells. (a) The positively charged protein transduction domain makes contact with the negatively charged outer membrane; (b) The protein translocates through the membrane in an unfolded state; (c) Once inside the cell, members of HSP 90 family refold the protein into an active conformation (S.Dowdy 2000). Runx3 is a member of Runx family that are critical regulators in inducing new tissues or determining cell fate. Runx 3 is expressed in normal gastrointestinal epithelial cells and has been suggested as a tumour suppressor involved in gastric cancer. The molecular mechanism of
Runx3 activity, however, is still poorly understood. In this paper, we synthesized a cell-permeable Runx3 terminal peptide for future study. By fusing the peptide with Tat transduction domain, we tried transducing the peptide under native conditions to preserve its structure and function, despite its difficulty mentioned above. The fusion protein has been applied into NIH/3T3 fibroblast and C2C12 myoblast cell lines. II. Materials and Methods. 1. Plasmid construction. The 90bp RUNX3 cDNA fragment from pT3-C41 was amplified by PCR and ligated into peGFP-C1 treated with XhoI and EcoRI. The eGFP cDNA and eGFP-RUNX3 fusion fragment were inserted into NcoI and EcoRI sites of pTAT (given by Dr. Steven Dowdy) to generate the in- frame expression constructs: pTAT-eGFP and pTAT- eGFP-Runx3 term., respectively.
2. Expression and purification of Tat-eGFP, Tat- eGFP-Runx3 term. fusion proteins. Two constructs were transformed into BL21(DE3)LysS by heat shock (420C in 50 seconds). The expression of proteins was induced by IPTG. The proteins were purified through Ni-NTA column using native lysis, wash and elution buffers (containing NaH2PO4, NaCl and imidazole, pH 8.0). These purification buffers were made according to Qiagen's instruction. 3. Invitro intracellular transduction assays. The mouse C2C12 myoblasts and NIH/3T3 fibroblasts were grown in DMEM medium supplemented with 20% (C2C12) or 10% FBS (NIH/3T3). Intracellular transduction assays were performed in 24-well plates (NUNC). Cells treated with Tat fusion proteins at various concentrations were quickly rinsed once in PBS after different time intervals and mounted in PBS under
coverslips. The fluorescence was detected and recored with fluorescent microscopy connecting with a computer that possesses a micrograph-recording software. III. Results and Discussion 1. Construction of expressing plasmids using pTAT plasmid. In order to amplify the 90bp fragment of RUNX3 terminal, specific primers were designed based on human RUNX3 sequence from NCBI gene bank (Figure 2B). The primers contain XhoI and EcoRI restriction sites to provide sites for the ligation of RUNX3 into peGFP-C1 plasmid at next step. The fusion of Runx3 with eGFP (enhanced green fluoresence protein) made possible the live observation of the transduction process, and is a sign that the protein's function is preserved during transduction process. Figure 2A shows the pTAT vector (~ 3kb) containing a T7 polymerase promoter, an ATG start codon, a 6 histidine
leader for Ni affinity purification, followed by the 11 amino-acid-TAT domain fused to the 5'-end of the HA tag. For the constructs, either eGFP or eGFP-RUNX3 term. was cloned into the multiple cloning site (NcoI/EcoRI) downstream of the HA sequence. The pTAT-cDNA plasmids were then transformed into DH5 bacterial strain which yields a high-copy plasmid number. The individual clones were isolated, the DNA sequence was confirmed by automated DNA sequencing. Figure 2. Plasmid construction. A- pTAT vector; B- specific primers for making Runx3 term. expressing construct; C- Two constructs that have been made.
2. Expression and purification of the Tat fusion proteins under native conditions. The constructed plasmids were transformed into high- expressing BL21(DE3)LysS bacterial strain. The expression was under the control of the IPTG-induced T7 promoter, in the presence of 1mM IPTG added to the baterial culture. Molecular weight of each protein is as follows: TAT-eGFP: 37 kDa; TAT-eGFP-Runx3 term.: 37.3 kDa. Purification of the proteins through a Ni-NTA affinity column was performed under native conditions (described above) to preserve their structure and function before introduced into the cells. The purification produced more than 95% pure yields at a final concentration of 0.2 to 6.5 mg/ml. (Figure 3).
Figure 3. SDS-polyacrylamide gel electrophoresis analysis of purified native proteins. (a): Fractions collected during purification procedure: M: protein marker; Ctrl: non-expressing bacteria; I(-): IPTG- non induced bacteria; I(+): IPTG-induced bacteria; St: crude bacterial lysate; Fl: flow-through; W1, W2: wash; E: eluate; E¬¬¬de: desalted eluate. (b): Distinguishing between pTAT-eGFP and pTAT-eGFP-Runx3 term. Samples were applied on 15% SDS-polyacrylamide gel and visualized by Coomassie blue staining. 3. Intracellular localization of the TAT-eGFP-Runx3 fusion protein in cultured cell lines. To access the transduction capacity, we added the Tat
fusion protein to the cultured media of NIH/3T3 and C cells at various concentrations of 10, 20, 50, 100, 190, 400 and 800ug/ml. The transduction process was observed under fluorescent microscopy. Intracellular green fluorescence was first detected in the cytoplasm of NIH/3T3 when cells were incubated with the protein at concentration of 190ug/ml in 12h. The number of cells uptaking the protein and intracellular green fluorescence were enhanced when the protein concentrations increased, indicating a concentration dependency for protein transduction which is consistent with previous studies, and thus, the ability to modulate intracellular concentration. At the protein concentration of 400ug/ml, 100% of cells showed green fluorescence. This demonstrates that the highest efficiency of protein transduction may be reached at the minimum concentration of 400 ug/ml. As a result, we were able to optimize the appropriate concentration of proteins to be applied efficiently for future experiments. (Figure 4). When we tested transduction assays on C2C12 mouse
myoblasts, transduction efficiency definitely fell down, showing the dependency of transduction efficiency on cell type. (Figure 5). Figure 4: Intracellular delivery of Tat-eGFP-Runx3 term. in NIH/3T3 fibroblasts. Fluoresence micrographs of NIH/3T3 treated with Tat- eGFP-Runx3 term. at (A) before transduced; (B) 190ug/ml; (C) 400ug/ml or (D) 800ug/ml and corresponding phase- constrast images (A', B', C' and D'). Cells were incubated for 12h, quickly washed and then mounted in PBS before examined.
Figure 5. Comparison of transduction efficiency between NIH/3T3 fibroblasts and C2C12 myoblasts. Fluoresence micrographs of NIH/3T3 treated with 800ug/ml of (A) Tat-eGFP or (C) Tat-eGFP-Runx3term.; C2C12 treated with 800ug/ml of (B) Tat-eGFP or (D) Tat- eGFP-Runx3term. Cells were incubated for 12h, quickly washed and then mounted in PBS before examined. IV. Conclusion. 1. Cell-permeable Runx3term. maining native structure were synthesized.
2. Highest efficiency of transduction into NIH/3T3 was obtained at the minimum protein concentration of 400ug/ml. 3. Transduction efficiency varies in two different cell lines studied. Acknowledgement. The authors would like to thank Dr. Kwang-Youl Lee and colleagues at Institute for Tumor Research, Chungbuk National University for their useful help and suggestions. Main references: 1. Green, M. and Loewenstein, P.M. (1988). "Autonomous functional domains of chemically synthesized human immunodeficiency virus TAT trans-activator protein." Cell 55: 1179-1188. 2. Frankel, A.D. and Pabo, C.O. (1988). "Cellular uptake of