İrfan TURHAN, Ülgen İlknur KONAK, Muharrem CERTEL

Proteinlerin Kromatografik Yöntemlerle Saflaştırılması

Kromatografi; kimya, biyoteknoloji, gıda ve tıp alanlarında analizde, izolasyonda ve saflaştırmada en yaygın kullanılan ayırma tekniğidir. Buna ilaveten, genellikle sanayide küçük ve büyük ölçekli üretimlerde prosesin bir parçası olarak kullanılmaktadır. Protein saflaştırma işlemi için yöntemlerin geliştirilmesi kimya, biyoloji, gıda ve tıp alanlarında yapılan birçok gelişme için önemli bir ön koşuldur. Protein saflaştırma, basit tek bir aşamada çöktürme işlemiyle veya büyük ölçekli onaylanmış proseslerle yapılmaktadır. Genel olarak, birden fazla aşamada yapılan saflaştırma işlemi istenilen saflığa ulaşmak için önemlidir. Saflaştırma işleminde kullanılacak olan yöntemler proteinlerin fiziksel, kimyasal ve biyolojik özellikleri dikkate alınarak seçilmektedir. Bu çalışmada, proteinlerin saflaştırılmasında kullanılan bazı kromatografik yöntemlerin temellerine ilişkin bilgiler verilmiştir.

Chromatographic Methods in Protein Purification

Chromatography is the most widely used separation technique in chemistry, biotechnology, food and medicine in analysis, isolation and purification. Additionally, it is commonly used in the industry as a component in the process of small and large-scale production. The development of methods for protein purification has been an essAkdeniz Üniversitesi, Mühendislik Fakültesi, Gıda Mühendisliği Bölümü, 07059, Antalya ential pre-requisite for many of the advancements in chemistry, biology, food and medicine. Protein purification varies from a simple one-step precipitation procedure to large scale validated processes. Generally, more than one purification step is necessary to reach the desired purity. Methods used in purification are selected based on the physical, chemical and biological properties of proteins. In this study, the fundamentals of some chromatographic methods used in purification of proteins are reviewed.

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Poole, C.F., 2000. Chromatography. http://www.kau.edu.sa/GetFile.aspx?id=108121&Ln g=AR&fn=CHROMATOGRAPHY.pdf
Kumpalume, P., Ghose, S., 2003. Chromatography: The High-Resolution Technique for Protein Separation. In: Isolation and Purification of Proteins, R. Hatti-Kaul and B. Mattiasson (Editors), Marcel Dekker, pp. 29-56, New York.
Hedhammar, M., Karlström, A.E., Hober, S., 2013. Chromatographic methods for protein purification. http://www.biotech.kth.se/courses/gru/courselist/BB 2040_ENG/ChromMethods.pdf
Doonan, S., Cutler, P., 2004. General Strategies. In: Protein Purification Protocols, P. Cutler (Editor), Humana Press, pp. 1-13, New Jersey.
Scopes, R.K., 2000. Protein Separation. In: Encyclopedia of Separation Science, M. Cooke and C.F. Poole (Editors), Academic Press, pp. 405-410, USA.
Sheehan, D., Fitzgerald, R., 2008. Ion-Exchange Chromatography. Handbook, R. Rapley and J.M. Walker (Editors), Humana Press, pp. 445-449, New Jersey.
Selkirk, C., 2004. Ion-exchange chromatography. Methods in Molecular Biology 244: 125-131.
Yamamoto, S., Miyagawa, E., 2000. Dynamic binding performance of large biomolecules such as ?-globulin, viruses and virus-like particles on various chromatographic supports. In: Bioseparation Engineering, I. Endo, T. Nagamune, S. Katoh and T. Yonemoto (Editors), Elsevier, pp. 81-86, New York.
Whitford, D., 2005. Protein expression, purification and characterization. In: Proteins: Structure and Function, John Wiley & Sons, pp. 313-346, England.
Kenney, A.C., 1992. Ion-Exchange Chromatography of Proteins. In: Methods in Molecular Biology, A. Kenney and S. Fowell (Editors), Humana Press, pp. 249-258, New Jersey.
Pohl, C.A., Stillian, J.R., Jackson, P.E., 1997. Factors controlling ion-exchange selectivity in suppressed ion chromatography. Journal of Chromatography A 789(1-2): 29-41.
Haddad, P.R., Jackson, P.E., 1990. Ion Chromatography: principles and applications. In: Journal of Chromatography Library-Volume 46, Elsevier, pp. 1-28, Amsterdam.
Yamamoto, S., Ishihara, T., 1999. Ion-exchange chromatography of proteins near the isoelectric points. Journal of Chromatography A 852(1): 31-36.
Hagel, L., Jagschies, G., Sofer, G., 2008. Separation Technologies. In: Handbook of Process Chromatography, Elsevier, pp. 81-122, London.
Haky, J.E., 2004. Ion-Exchange Buffers. In: Encyclopedia of Chromatography, J. Cazes (Editor), Marcel Dekker, pp. 794-795, New York.
Gooding, K.M., 2004. Ion Exchange: Mechanism and Factors Affecting Separation. In: Encyclopedia of Chromatography, J. Cazes (Editor), Marcel Dekker, pp. 790-793, New York.
McCue, P.P., Shetty, K., 2006. Principles of Biochemistry and Molecular Biology. In: Food Biotechnology, K. Shetty, A. Pometto and R. E. Levin (Editors), CRC Press, pp. 19-32, Boca Raton.
Anonymous, 2002. Ion Exchange Chromatography. In: Protein Separation (Handbook Collection Amersham), pp. 498-654.
Sadana, A., 1998. Protein Inactivations during Chromatographic Methods of Separation. In: Bioseparation of Proteins, S. Ahuja (Editor), Academic Press, pp. 135-176, New York.
Roe, S., 1989. Separation Based on Structure. In: Protein Purification Methods (a practical approach), E.L.V. Harris and S. Angal (Editors), Oxford University Press, pp. 175-242, New York.
Grund, E., 1998. Hydrophobic Interaction Chromatography of Proteins. In: Bioseparation and Bioprocessing, G. Subramanian (Editor), WileyVCH, pp. 65-87, New York.
Gooding, K.M., 2004. Hydrophobic Interaction Chromatography. In: Encyclopedia of Chromatography, J. Cazes (Editor), Marcel Dekker, pp. 751-754, New York.
Lienqueo, M.E., Mahn, A., Vásquez, L., Asenjo, J.A., 2003. Methodology for predicting the separation of proteins by hydrophobic interaction chromatography and its application to a cell extract. Journal of Chromatography A 1009: 189-196.
Hearn, M.T.W., 2002. Reversed-Phase and Hydrophobic Interaction Chromatography of Proteins and Peptides. In: HPLC of Biological Macromolecules, K.M. Gooding and F.E. Regnier (Editors), Marcel Dekker, pp. 99-245, New York
Anonymous, 2002. Hydrophobic Interaction Chromatography. In: Protein Separation (Handbook Collection Amersham), pp. 2-104.
O'Farrell, P.A., 2008. Hydrophobic Interaction Chromatography. In: Molecular Biomethods Handbook, R. Rapley and J.M. Walker (Editors), Humana Press, pp. 461- 467, New Jersey.
Jennissen, H.P., 2000. Hydrophobic Interaction Chromatography. In: Encyclopedia of Separation Science, M. Cooke and C.F. Poole (Editors), Academic Press, pp. 265-272, USA.
Queiroz, J.A., Tomaz, C.T., Cabral, J.M.S., 2001. Hydrophobic proteins. Journal of Biotechnology 87: 143-159.
Cartwright, T., 2001. Protein purifications from mammalian cell culture. In: Protein Purification Applications, S. Roe (Editor), Oxford University Press, pp. 49-71, New York.
Zou, H., Luo, Q., Zhou, D., 2001. Affinity membrane chromatography for the analysis and purification of proteins. Journal of Biochemical and Biophysical Methods 49: 199-240.
Wilchek, M., Chaiken, I., 2000. An Overview of Affinity Chromatography. In: Affinity Chromatography-Methods and Protocols, P. Bailon, G.K. Ehrlich, Wen-Jian Fung and W. Berthold (Editors), Humana Press, pp. 1-6, New Jersey.
Hage, D.S., 2006. Handbook of Affinity Chromatography, D.S. Hage and J. Cazes (Editors), CRC Pres, p 856, Boca Raton. (Editors), CRC Pres, p 856, Boca Raton.
Anonymous, 2002. Affinity Chromatography. In: Protein Separation (Handbook Collection Amersham), pp. 230-389.
Ostrove, S., 1990. Affinity Chromatography: General Methods. In: Methods in Enzymology, J.N. Abelson and M.I. Simon (Editors), Academic Press, pp. 357-371, New York.
Hage, D.S., Clarke, W., 2004. Affinity Chromatography: An Overview. In: Encyclopedia of Chromatography, J. Cazes (Editor), Marcel Dekker, pp. 40-43, New York.
Hage, D.S., 1999. Affinity Chromatography: A Review of Clinical Applications. Clinical Chemistry, 45, 593-615.
Rosenberg, I.M. 2005. Affinity Chromatography. In: Protein Analysis and Purification (Benchtop Techniques), Birkhäuser, pp. 363-381, Basel.
Firer, M.A., 2001. Efficient elution of functional proteins in affinity chromatography. Journal of Biochemical and Biophysical Methods 49: 433-442.
Anonymous, 2002. Gel Filtration. In: Protein Separation (Handbook Collection Amersham), pp. 106-227.
Cutler, P., 2004. Size-Exclusion Chromatography. In: Protein Purification Protocols, P. Cutler (Editor), Humana Press, pp. 239-252, New Jersey.
Rosenberg, I.M., 2005. Gel Filtration Chromatography. In: Protein Analysis and Purification (Benchtop Techniques), Birkhäuser, pp. 326-338, Basel.
Winzor, D.J., 2003. Analytical exclusion chromatography. Journal of Biochemical and Biophysical Methods 56: 15-52.