To grow protein crystals have been so far recognized to be a not easy task, due to the high level of complexity of bio-macromolecules. Furthermore, once crystals have been obtained, their characteristics, determined heavily from the crystallization kinetic, may be crucial in determining their effective applicability. Indeed, while protein crystallization were first utilised for purification purposes, the subsequently advancements in the field of X-ray analysis, have opened new extraordinary opportunity for structure determination. Today, protein crystallization has been mainly developed for structure resolution, giving a fundamental contribution to the field of rationale drug design. However, this can be possible only by producing single crystals with adequate size (about 100 ?m) and with high internal order [1]. A more recent trend in protein crystallization, relies on the production of cross linked enzyme crystals (CLECs). Such materials generally demonstrated enhanced stability against denaturation by heat, organic solvents and proteolysis, and, depending on some parameters, no loss or even increase in enzymatic activity in the crystalline state in respect to the corresponding soluble protein. However, a fundamental characteristics for CLECs is the control of their size [2]. To produce enzyme crystals by an opportune technique that allows to control the final properties of the products, would be an essential improvement in the overall field of life sciences. Membrane crystallization has been recently proposed as a new technique for producing protein crystals with enhanced crystallization kinetic, by utilising the polymeric membrane surface as a promoter of heterogeneous crystallization, without detrimental effects or even with improvements, on the overall crystallographic quality of the crystals. Primary tests were conducted using lysozyme as model protein [3,4]. Aim of the present work is to better understand and control, by acting on the operative parameters involved in the process, the crystallization kinetic of a model protein belonging to the enzyme family of serine proteases. In fact, in our purposes, this would be a fundamental contribution for developing a protein, and more specifically enzyme, crystallization technique, for producing crystals with adequate characteristics, in terms of size, size distribution, internal quality, depending on the specific application required, which may be to produce single crystals for structural investigations, or crystalline materials as the first step in the formulation of CLECs. Experimental results on the crystallization kinetic of trypsin from bovine pancreas, confirmed the effectiveness of the novel membrane-based method. Moreover, X-ray analysis on the obtained crystals, confirmed the crystallographic quality of the enzyme crystals produced. [1] A. McPherson, Methods 34 (2004) 254-265. [2] A.L. Margolin, M.A. Navia, Angew. Chem. Int. ed. 40 (2001) 2204-2222. [3] G. Di Profio, E. Curcio, A. Cassetta, D. Lamba, E. Drioli, J. Crys. Growth 257 (2003) 359-369. [4] E. Curcio, S. Simone, G. Di Profio, E. Drioli, A. Cassetta, D. Lamba, J. Mem. Sci., in press.
Membrane-based techniques for enzymes crystallization: a possible application in bio-pharmaceutical industry
G Di Profio;
2004
Abstract
To grow protein crystals have been so far recognized to be a not easy task, due to the high level of complexity of bio-macromolecules. Furthermore, once crystals have been obtained, their characteristics, determined heavily from the crystallization kinetic, may be crucial in determining their effective applicability. Indeed, while protein crystallization were first utilised for purification purposes, the subsequently advancements in the field of X-ray analysis, have opened new extraordinary opportunity for structure determination. Today, protein crystallization has been mainly developed for structure resolution, giving a fundamental contribution to the field of rationale drug design. However, this can be possible only by producing single crystals with adequate size (about 100 ?m) and with high internal order [1]. A more recent trend in protein crystallization, relies on the production of cross linked enzyme crystals (CLECs). Such materials generally demonstrated enhanced stability against denaturation by heat, organic solvents and proteolysis, and, depending on some parameters, no loss or even increase in enzymatic activity in the crystalline state in respect to the corresponding soluble protein. However, a fundamental characteristics for CLECs is the control of their size [2]. To produce enzyme crystals by an opportune technique that allows to control the final properties of the products, would be an essential improvement in the overall field of life sciences. Membrane crystallization has been recently proposed as a new technique for producing protein crystals with enhanced crystallization kinetic, by utilising the polymeric membrane surface as a promoter of heterogeneous crystallization, without detrimental effects or even with improvements, on the overall crystallographic quality of the crystals. Primary tests were conducted using lysozyme as model protein [3,4]. Aim of the present work is to better understand and control, by acting on the operative parameters involved in the process, the crystallization kinetic of a model protein belonging to the enzyme family of serine proteases. In fact, in our purposes, this would be a fundamental contribution for developing a protein, and more specifically enzyme, crystallization technique, for producing crystals with adequate characteristics, in terms of size, size distribution, internal quality, depending on the specific application required, which may be to produce single crystals for structural investigations, or crystalline materials as the first step in the formulation of CLECs. Experimental results on the crystallization kinetic of trypsin from bovine pancreas, confirmed the effectiveness of the novel membrane-based method. Moreover, X-ray analysis on the obtained crystals, confirmed the crystallographic quality of the enzyme crystals produced. [1] A. McPherson, Methods 34 (2004) 254-265. [2] A.L. Margolin, M.A. Navia, Angew. Chem. Int. ed. 40 (2001) 2204-2222. [3] G. Di Profio, E. Curcio, A. Cassetta, D. Lamba, E. Drioli, J. Crys. Growth 257 (2003) 359-369. [4] E. Curcio, S. Simone, G. Di Profio, E. Drioli, A. Cassetta, D. Lamba, J. Mem. Sci., in press.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
