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

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

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 C2C12myoblast 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: eGFP and

pTAT-eGFP-Runx3 term., respectively

2 Expression and purification of 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

the TAT protein from human immunodeficiency virus." Cell 55: 1189-1193

3 Paul A Wender, D.J.M., Kanaka Pattabiraman, Erin T Pelkey, Lawrence Steinman (2000) "The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: peptoid molecular transporter." PNAS 97: 13003-

6 Christine Schneider, L S.-L., Elmar Nimmesgern,

Ouathek Ouerfelli, Samuel Danishefsky, Neal Rosen, F Ulrich Hartl (1996) "Pharmacologic shifting of a balance between protein refolding and degradation mediated by Hsp90" Proc Natl Acad Sci USA 93: 14536-14541

oa học: GS Lê Đình Lương