Tan Yan Peng
(Department of Chemical and Environmental Engineering, Institute of Bioscience, University Putra Malaysia)
,
Ling Tau Chuan
(Department of Process & Food Engineering, Faculty of Engineering, Institute of Bioscience, University Putra Malaysia)
,
Yusoff Khatijah
(Institute of Bioscience, Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia)
,
Tan Wen Siang
(Institute of Bioscience, Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia)
,
Tey Beng Ti
(Department of Chemical and Environmental Engineering, Institute of Bioscience, University Putra Malaysia)
In the present study, the performances of conventional purification methods, packed bed adsorption (PBA), and expanded bed adsorption (EBA) for the purification of the nucleocapsid protein (NP) of Newcastle disease virus (NDV) from Escherichia coli homogenates were evaluated. The conventional method...
In the present study, the performances of conventional purification methods, packed bed adsorption (PBA), and expanded bed adsorption (EBA) for the purification of the nucleocapsid protein (NP) of Newcastle disease virus (NDV) from Escherichia coli homogenates were evaluated. The conventional methods for the recovery of NP proteins involved multiple steps, such as centrifugation, precipitation, dialysis, and sucrose gradient ultracentrifugation. For the PBA, clarified feedstock was used for column loading, while in EBA, unclarified feedstock was used. Streamline chelating immobilized with $Ni^{2+}$ ion was used as an affinity ligand for both PBA and EBA. The final protein yield obtained in conventional and PBA methods was $1.26\%$ and $5.56\%$, respectively. It was demonstrated that EBA achieved the highest final protein yield of $9.6\%$ with a purification factor of 7. Additionally, the total processing time of the EBA process has been shortened by 8 times compared to that of the conventional method.
In the present study, the performances of conventional purification methods, packed bed adsorption (PBA), and expanded bed adsorption (EBA) for the purification of the nucleocapsid protein (NP) of Newcastle disease virus (NDV) from Escherichia coli homogenates were evaluated. The conventional methods for the recovery of NP proteins involved multiple steps, such as centrifugation, precipitation, dialysis, and sucrose gradient ultracentrifugation. For the PBA, clarified feedstock was used for column loading, while in EBA, unclarified feedstock was used. Streamline chelating immobilized with $Ni^{2+}$ ion was used as an affinity ligand for both PBA and EBA. The final protein yield obtained in conventional and PBA methods was $1.26\%$ and $5.56\%$, respectively. It was demonstrated that EBA achieved the highest final protein yield of $9.6\%$ with a purification factor of 7. Additionally, the total processing time of the EBA process has been shortened by 8 times compared to that of the conventional method.
* AI 자동 식별 결과로 적합하지 않은 문장이 있을 수 있으니, 이용에 유의하시기 바랍니다.
제안 방법
coli homogenates with EBA has not been reported elsewhere in the literature. In this work, we investigated the performance of three different modes of operations employed for the recovery ofrecombinant NP protein, namely conventional methods (combination of precipitation and ultracentrifugation), packed bed adsorption (PBA) and EBA. The final yield and purity of the product and processing times of these three methods are compared.
대상 데이터
Streamline 25 Column (ID = 25 mm), XK 16/20 Column (ID = 16 mm) and Streamline chelating were purchased from Amersham Bioscience (Uppsala, Sweden). The adsorbent has a mean particle density of 1.
성능/효과
, 1999) and can be operated at room temperature. In conclusion, EBA is an attractive alternative to the conventional purification method, with the advantages of less unit operation involved, shorter processing time, higher yield, lower capital investment, and lower operational costs.
, 1999). It was demonstrated that a single-step purification using EBA recorded a 7.6-fdld higher final protein yield compared to the conventional method. Fig.
참고문헌 (25)
Ameskamp, N., C. Priesner, J. Lehmann, and D. Lutkemeyer. 1999. Pilot scale recovery of monoclonal antibodies by expanded bed ion exchange adsorption. Bioseparation 8, 169-88
Anspach, F.B., D. Curbelo, R. Hartmann, G. Garke, and W.D. Deekwer. 1999. Expanded-bed chromatography in primary protein purification. J. Chromatogr. A 865, 129-144
Batt, B.C., V.M. Yabannavar, and V. Singh. 1995. Expanded bed adsorption process for protein recovery from whole mammalian cell culture broth. Bioseparation 5, 41-52
Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254
Chaga, G.S. 2001. Twenty-five years of immobilized metal ion affinity chromatography: past, present and future. J. Biochem. Biophys. Methods 49, 313-334
Chang, Y.K. and H.A. Chase. 1996. Ion exchange purification of G6PDH from unclarified yeast cell homogenates using expanded bed adsorption. Biotechnol. Bioeng. 49, 204-216
Ersson, B., L. Ryden, and J.-C. Janson. 1998. Introduction to protein purification, p. 3 - 40. In J-C. Janson, L. Ryden (eds.), Protein Purification: Principles, High-Resolution Methods, and Applications. John Wiley & Sons, New York
Hu, Y.C., C. T. Tsai, Y.C. Chung, J.T. Lu, and J.T. Hsu. 2003. Generation of chimeric baculovirus with histidine-tags displayed on the envelope and its purification using immobilized metal affinity chromatography. Enzyme Microb. Technol. 33, 445-452
Kho, C.L., W.S. Tan, and K. Yusoff. 2001. Production of the nucleocapsid protein of Newcastle disease virus in Escherichia coli and its assembly into ring- and nucleocapsid-like particles. J. Microbiol. 39, 293-299
Ling, T.C. 2002. Ph. D. thesis. The University of Birmingham, UK
Ling, T.C., C.K. Long, W.S. Tan, B.T. Tey, A. Ariff, W.M. Wan Abdullah. 2004. Purification of filamentous bacteriophage M13 by expanded bed anion exchange chromatography. J. Micorbiol. 42, 22-232
Noppe, W., I. Hanssens, and M.D. Cuyper. 1996. Simple two step procedure for the preparation of highly active pure equine milk lysozyme. J. Chromatogr. A 719, 327-331
Rabu, A., W.S. Tan, C.L. Kho, A.R. Omar, and K. Yusoff. 2002. Chimeric Newcastle disease virus nucleocapsid with parts of its viral haemagglutinin-neuraminidase and fusion proteins. Acta Virol. 46, 211-217
Sumi, A., K. Okuyama, K. Kobayashi, W. Ohtani, T. Ohmura, and K. Yokoyama. 1999. Purification of recombinant human serum albumin efficient purification using STREAMLINE. Bioseparation 8, 195-200
Thommes, J., A. Bader, M. Halfar, A. Karau, and M-R Kula. 1996. Isolation of monoclonal antibodies from cell containing hybridoma broth using a protein A. coated adsorbent in expanded beds. J. Chromatogr. A 752, 111-122
Thommes, J., M. Halfar, S. Lenz, M.-R. Kula. 1995. Purification of monoclonal antibodies from whole hybridoma fermentation broth by fluidized bed adsorption. Biotechnol. Bioeng. 45, 205-211
Willoughby, N.A., T. Kirschner, M.P. Smith, R. Hjorth, and N.J. Titchener-Hooker. 1999. Immobilized metal affinity chromatography purification of alcohol dehydrogenase from Baker's yeast using an expanded bed adsorption system. J. Chromatogr. A 840, 195-204
Xiang, H., R. Wynn, L-H. T. Nguyen, O.H. Ross, D.P. Ahrens, K.T. O'Neil, G.F. Hollis, and D.R. Patrick. 2002. Immobilized metal-ion affinity chromatography of recombinant Fab protein OPG C11 in the presence of EDTA-Mg (II). J. Chromatogr. A 978, 153-164
※ AI-Helper는 부적절한 답변을 할 수 있습니다.