선행연구에서 카스파제-3 효소가 DEVD 기질을 완전히 절단하는 반면 IETD 기질은 DEVD의 약 50%정도만을 부분적으로 분해한다는 사실을 보고하였다. 본 연구에서는 정제된 폴리-단백질이 카스파제-3 단백질 분해효소의 기질에 따른 차별적인 분해활성에 의해 프로세싱 되는 양상을 분석하였다. 모델 단백질로서 GST, MBP, RFP 세 종류의 단백질을 DEVD 및 IETD 펩타이드로 연결시킨 폴리-단백질을 제작하였으며, 폴리-단백질은C-말단에 6개의 히스티딘태그가 결합되도록 클로닝 되었다. IMAC크로마토그래피를 이용하여 분리 및 정제된 재조합 단백질은 SDS-PAGE 분석을 통해 분자량이 약 93 kDa으로 나타났으며, 카스파제-3 효소의 처리에 의해 각각 MBP:RFP, MBP 그리고 GST 3종류의 단백질 절편으로 절단 및 분리되었다. 이 연구를 통해 단백질 분해효소와 기질 간의 반응성의 차이를 이용하여 폴리-단백질을 정량적으로 프로세싱 할 수 있다는 예를 보여주었다.
선행연구에서 카스파제-3 효소가 DEVD 기질을 완전히 절단하는 반면 IETD 기질은 DEVD의 약 50%정도만을 부분적으로 분해한다는 사실을 보고하였다. 본 연구에서는 정제된 폴리-단백질이 카스파제-3 단백질 분해효소의 기질에 따른 차별적인 분해활성에 의해 프로세싱 되는 양상을 분석하였다. 모델 단백질로서 GST, MBP, RFP 세 종류의 단백질을 DEVD 및 IETD 펩타이드로 연결시킨 폴리-단백질을 제작하였으며, 폴리-단백질은C-말단에 6개의 히스티딘 태그가 결합되도록 클로닝 되었다. IMAC 크로마토그래피를 이용하여 분리 및 정제된 재조합 단백질은 SDS-PAGE 분석을 통해 분자량이 약 93 kDa으로 나타났으며, 카스파제-3 효소의 처리에 의해 각각 MBP:RFP, MBP 그리고 GST 3종류의 단백질 절편으로 절단 및 분리되었다. 이 연구를 통해 단백질 분해효소와 기질 간의 반응성의 차이를 이용하여 폴리-단백질을 정량적으로 프로세싱 할 수 있다는 예를 보여주었다.
In this study, it is reported that a large polyprotein can be stoichiometrically cleaved by the use of caspase-3-dependent proteolysis. Previously, it has been shown that the proteolytic IETD motif was partially processed when treated with caspase-3, while the DEVD motif was completely cleaved. The ...
In this study, it is reported that a large polyprotein can be stoichiometrically cleaved by the use of caspase-3-dependent proteolysis. Previously, it has been shown that the proteolytic IETD motif was partially processed when treated with caspase-3, while the DEVD motif was completely cleaved. The cleavage efficiency of the DEVD-based substrate was approximately 2.0 times higher than that of the IETD substrate, in response to caspase-3. Based on this, 3 protein genes of interest were genetically linked to each other by adding two proteolytic cleavage sequences, DEVD and IETD, for caspase-3. Particularly, glutathione-S transferase (GST), maltose binding protein (MBP), and red fluorescent protein (RFP) were chosen as model proteins due to the variation in their size. The expressed polyprotein was purified by immobilized metal ion affinity chromatography (IMAC) via a hexa-histidine tag at the C-terminal end, showing 93 kDa of a chimeric GST:MBP:RFP fusion protein. In response to caspase-3, cleavage products, such as MBP:RFP (68 kDa), MBP (42 kDa), RFP (26 kDa), and GST (25 kDa), were separated from a large precursor GST:MBP:RFP (93 kDa) via SDS-PAGE. The results obtained from this study indicate that a multi-protein can be stoichiometrically produced from a large poly-protein by using proteolytic recognition motifs, such as DEVD and IETD tetra-peptides, for caspase-3.
In this study, it is reported that a large polyprotein can be stoichiometrically cleaved by the use of caspase-3-dependent proteolysis. Previously, it has been shown that the proteolytic IETD motif was partially processed when treated with caspase-3, while the DEVD motif was completely cleaved. The cleavage efficiency of the DEVD-based substrate was approximately 2.0 times higher than that of the IETD substrate, in response to caspase-3. Based on this, 3 protein genes of interest were genetically linked to each other by adding two proteolytic cleavage sequences, DEVD and IETD, for caspase-3. Particularly, glutathione-S transferase (GST), maltose binding protein (MBP), and red fluorescent protein (RFP) were chosen as model proteins due to the variation in their size. The expressed polyprotein was purified by immobilized metal ion affinity chromatography (IMAC) via a hexa-histidine tag at the C-terminal end, showing 93 kDa of a chimeric GST:MBP:RFP fusion protein. In response to caspase-3, cleavage products, such as MBP:RFP (68 kDa), MBP (42 kDa), RFP (26 kDa), and GST (25 kDa), were separated from a large precursor GST:MBP:RFP (93 kDa) via SDS-PAGE. The results obtained from this study indicate that a multi-protein can be stoichiometrically produced from a large poly-protein by using proteolytic recognition motifs, such as DEVD and IETD tetra-peptides, for caspase-3.
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문제 정의
The method of ion exchange can be also combined with gel filtration and affinity chroma- tography in a single column, allowing for the effective purifi- cation of target proteins varying in molecular weight as well as the value of the isoelectric point (pI). This work showed the possibility of muti-protein production, based on cas- pase-3-dependent cleavage fragments from a single poly- protein in denaturing SDS-PAGE separation. Thus, further studies are required for the removal of protease used, and the successful isolation of proteolytic cleavage products as a biologically active form.
제안 방법
In order to clone the chimeric GST:MBP:RFP harboring DEVD and IETD motifs, the full-length genes encoding for MBP and RFP were amplified with the 5‘ primers (CG GGA TCC GAT GAG GTG GAT GGA GAA TTC ATG AAA ACT GAA GAA GG, CCC AAG CTT ATG GTG CGC TCC TCC AAG) and the 3’ primers (CCC AAG CTT TCC ATC GGT CTC GAT GTC GAC AGT CTG CGC GTC TTT CAG, CCG CTC GAG CAG GAA CAG GTG GTG GCG), respectively, via the polymerase chain reaction (PCR). The 5’and 3’ termi- ni were designed to harbor the BglII-BamHI, EcoRI-Sall, HindⅢ-XhoI restriction enzyme cleavage sites, respectively.
Viral polyprotein must be processed by protease to produce functional cleavage products from a single precussor polypeptide. This work began with the idea that the feature of polyprotein cleavage can be genet- ically mimicked by inserting the cleavage sites between the target protein genes, and the mode of polyprotein processing can be used to obtain multi-protein from a single recombi- nant polyprotein. In this study, caspase-3 as a protease re- sponsible for the cleavage was chosen, because its recog- nition tetrapeptides are well extensively studied [12].
이론/모형
The gel was stained with Coomassie’s Brilliant Blue R250. Protein concentrations were determined by the Brad- ford method, using bovine serum albumin as a standard. For the quantification of SDS-PAGE data, the band densi- tometry analysis was performed with Image J software.
성능/효과
2A). After IPTG induction, an extra protein band with an approximate molecular weight of 93 kDa, which is consistent with the expected mass of GST:MBP:RFP predicted using ExPASy Proteomics tools (www.expasy.org), was found only in the E. coli cultures harboring pGST:MBP:RFP plasmid, and most of the expressed target protein has appeared in the soluble fraction, and has seldom existed in inclusion bodies (data not shown). In order to obtain chimeric GST:MBP:RFP precussor molecule, the recombinant GST:MBP:RFP poly- protein was purified using by metal chelate affinity chroma-tography performed in IDA Excellose column charged with Ni2+ ions.
For the quantification of SDSPAGE data, the band densitometry analysis was per- formed with Image J software. As the amount of GST:MBP: RFP precussor polyprotein without caspase-3 was set at 100%, the relative level of MBP:RFP, MBP, RFP, and GST was approximately 27%, 10%, 12%, and 51%, respectively (Fig. 3A and 3B). Considering that approximately 50% of IETD substrates were partially cleaved in comparison with DEVD substrates, the expected relative amounts of the proc- essed products come to 25.
3A and 3B). Considering that approximately 50% of IETD substrates were partially cleaved in comparison with DEVD substrates, the expected relative amounts of the proc- essed products come to 25.0% for MBP:RFP, 12.5% for MBP, 12.5% for RFP, and 50.0% for GST. Therefore, our results are in good agreement with the theoretical expectation.
3A, the GST:MBP:RFP polyprotein was fully processed in 30 minutes after treatment with active cas- pase-3. The resulting cleavage products including MBP:RFP (68 kDa), MBP (42 kDa), RFP (26 kDa), and GST (25 kDa) were successfully separated via SDS-PAGE as indicated by the fragmented products, while no cleavage of GST:MBP: RFP was observed in response to mutant caspase-3, verifying the critical role of 163 cysteine of caspase-3 in its proteolytic activity. The finding obtained from this study shows the ad- vantages of the polyprotein-based muti-protein separation such as simplicity and rapidity of use.
Following treatment with caspase-3, four cleavage subunits such as MBP:RFP (68 kDa), MBP (42 kDa), RFP (26 kDa), and GST (25 kDa) were generated from a sin- gle polypeptide GST:MBP:RFP (93 kDa). These results showed that multi-protein can be stoichiometrically produced from a single recombinant polyprotein associated with the proteo- lytic recognition motifs, DEVD and IETD, showing cleavage preference for caspase-3.
후속연구
This work showed the possibility of muti-protein production, based on cas- pase-3-dependent cleavage fragments from a single poly- protein in denaturing SDS-PAGE separation. Thus, further studies are required for the removal of protease used, and the successful isolation of proteolytic cleavage products as a biologically active form.
참고문헌 (13)
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