Trypsin crystallization by membrane-based techniques.
To grow protein crystals is not an easy task; moreover, if we need to grow protein crystals with controlled shape, size, and size distribution, depending on their application, the mission becomes even harder. Membrane crystallization has been recognized as an interesting tool for growing protein crystals with enhanced crystallization kinetics, both in static and in forced solution flow configuration, without detrimental effects on crystal quality. In the present work, we have studied the membrane crystallization process of benzamidine inhibited trypsin from bovine pancreas (BPT), with ammonium sulphate (dissolved in Tris-HCl buffer, 0.1 M, pH 8.5), as precipitant agent. We have demonstrated that, by using the membrane crystallization technique, BPT crystals can be obtained in 24-48 h, in static configuration, and in 4-7 days, in a forced solution flow system, depending on the experimental conditions. Furthermore, the kinetics of BPT crystallization have been modulated, to control the morphological characteristics of the crystals produced, by an accurate selection of the operative parameters involved in the process. The active membrane surface and the flow rate of extraction solvent in quiescent configuration, and the solution velocity in forced convection solution experiments, were the parameters investigated. In this respect, membrane crystallization techniques have been assessed as an interesting way for growing proteins, and more specifically enzyme crystals, with high control on the final properties of the crystalline material produced, with potential fundamental implication in the field of structural biology and biotechnology.
Di Profio
Gianluca
G
Institute on Membrane Technology (ITM-CNR), c/o University of Calabria, Via P. Bucci, CUBO 17/C I-87030, Arcavacata di Rende (CS), Italy. g.diprofio@itm.cnr.it
Curcio
Efrem
E
Drioli
Enrico
E
D000818:Animals; D002417:Cattle; D003460:Crystallization / Q000295:instrumentation / Q000379:methods*; D008567:Membranes, Artificial; D014357:Trypsin / Q000737:chemistry*
D008567:Membranes, Artificial; D014357:Trypsin