Abstract
Seeding is a versatile method for optimizing crystal growth. Coupling this technique with capillary counter diffusion crystallization enhances the size and diffraction quality of the crystals. In this article, crystals for organic solvent-tolerant recombinant elastase strain K were successfully produced through microseeding with capillary counter-diffusion crystallization. This technique improved the nucleation success rate with a low protein concentration (3.00 mg/mL). The crystal was grown in 1 M ammonium phosphate monobasic and 0.1 M sodium citrate tribasic dihydrate pH 5.6. The optimized crystal size was 1 × 0.1 × 0.05 mm3. Elastase strain K successfully diffracted up to 1.39 Å at SPring-8, Japan, using synchrotron radiation for preliminary data diffraction analysis. The space group was determined to be monoclinic space group P1211 with unit cell parameters of a = 38.99 Ǻ, b = 90.173 Å and c = 40.60 Å.
Highlights
Crystallographers have developed many protein crystallization methods
Elastase strain K was purified through a series of steps conducted in a cold room to avoid autolysis
The purity of recombinant elastase strain K was examined using native-PAGE (Figure 1b), where the protein migrated as a single band
Summary
Crystallographers have developed many protein crystallization methods. More than 20 techniques have been reported; the most commonly used techniques are microbatch, counter diffusion, dialysis and hanging and sitting drop [1,2,3,4]. The method developed from the crystallization phase diagram is the seeding method [5,6], in which authors attempted to decouple nucleation and growth Another crystallization method, the counter-diffusion method, provides better crystallization conditions for growing crystals [7]. The counter-diffusion method, provides better crystallization conditions for growing crystals [7] These methods were developed for enhancing the quality and size of the crystals for atomic structure determination. The purification results show that this bacterium produces three protease types One of these proteases was highly similar to the elastase from P. aeruginosa. Our research will focus on recombinant elastase strain K crystallization and structure determination to discern its structural features as an organic solvent-stable enzyme. We present the crystallization technique used to produce high-diffraction quality crystals for elastase strain K and preliminary X-ray crystallographic data
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