The production of protein crystals with well defined characteristics has become a crucial task concerning the overall field of life sciences. Indeed, while first utilised for purification purposes, protein crystallization has been mainly developed in the last 50 years for structure determination, at atomic resolution, of proteins of therapeutic interest, thank to the development of X-ray diffraction techniques. However, the main obstacle to structure determination, is to prepare protein crystals with opportune size (usually higher than 100 ?m) and, with no minor importance, having high internal order [1]. Over the last years, a new field of application of protein, in particular enzymes, crystallization for biological catalysis have been developed. This is due to the discovery that some cross linked enzymes crystals (CLECs) demonstrated high 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 proteins. This allows to use enzymes, in a stable formulation, in chemical catalysis, where the harsh environment has prevented by now their utilisation directly in homogeneous phase. The fundamental characteristics for CLECs is their production with uniform shape and size. To grow crystals with high surface to volume ratio (small crystals), but easily handling for catalyst recovery and recycling (no too small crystals), would be an essential requirements for the yield of their applications [2]. Catalysts monodispersity is furthermore a well known requisite for high efficiency in heterogeneous catalysis. Recently, a new membrane-based technique have been proposed for protein crystallization. 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, were observed on primary tests conducted using lysozyme as model protein, in both static and forced solution configuration [3,4]. In the present work, membrane crystallization technique have been utilised to better 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 shape, 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. We present our results on the crystallization kinetic of trypsin from bovine pancreas, grown using membrane crystallization technique in both static and dynamic configurations, in order to confirm the effectiveness of the novel membrane-based method.
Preparation of enzyme crystals with controlled size and shape by using membrane crystallization technique
G Di Profio;
2005
Abstract
The production of protein crystals with well defined characteristics has become a crucial task concerning the overall field of life sciences. Indeed, while first utilised for purification purposes, protein crystallization has been mainly developed in the last 50 years for structure determination, at atomic resolution, of proteins of therapeutic interest, thank to the development of X-ray diffraction techniques. However, the main obstacle to structure determination, is to prepare protein crystals with opportune size (usually higher than 100 ?m) and, with no minor importance, having high internal order [1]. Over the last years, a new field of application of protein, in particular enzymes, crystallization for biological catalysis have been developed. This is due to the discovery that some cross linked enzymes crystals (CLECs) demonstrated high 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 proteins. This allows to use enzymes, in a stable formulation, in chemical catalysis, where the harsh environment has prevented by now their utilisation directly in homogeneous phase. The fundamental characteristics for CLECs is their production with uniform shape and size. To grow crystals with high surface to volume ratio (small crystals), but easily handling for catalyst recovery and recycling (no too small crystals), would be an essential requirements for the yield of their applications [2]. Catalysts monodispersity is furthermore a well known requisite for high efficiency in heterogeneous catalysis. Recently, a new membrane-based technique have been proposed for protein crystallization. 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, were observed on primary tests conducted using lysozyme as model protein, in both static and forced solution configuration [3,4]. In the present work, membrane crystallization technique have been utilised to better 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 shape, 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. We present our results on the crystallization kinetic of trypsin from bovine pancreas, grown using membrane crystallization technique in both static and dynamic configurations, in order to confirm the effectiveness of the novel membrane-based method.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
