| Summary: | 碩士 === 中興大學 === 生物化學研究所 === 95 === Xcc (Xanthomonas campestris pv .campestris) is a Gram-negative, plant-pathogenic bacterium that hosts mainly on crucifers. When infected by this bacterium, hosts show syndrome of marginal leaf chlorosis, or rotten leafs. Owing to high correlation between protein function and its structure, structural approach might be a feasible way that helps us understand more about protein function and its acting mechanism. For this reason, several functional proteins from Xcc were chosen as targets for structural studies. In the last two years, eight functional proteins were tested for their own conditions for expression, purification, and crystallization. To get reasonable amount of soluble protein, different expression strategies such as changing induction conditions, fusion protein, and hosts, etc were undertaken. Crystallization screening was followed by purification process.
After several conditions were tested, five out of the eight target proteins we were still unable to obtain enough material for crystallization screening progress. XC2428 (deoxycytidine triphosphate deaminase) was successfully expressed as a soluble protein by fusing MBP to its N-terminus to improve its solubility. It is, unfortunately, hard to be purified due to the rapid self-degradation, which make it difficult to get good protein crystals. XC20 (truncated IS1477 transposase) shows significant improvement in solubility after MBP fusion, but almost all XC20 protein precipitated after MBP removal by TEV protease cleavage. Simarily, although XC663 (DNA repair system specific for alkylated DNA) can achieve good solubility after MBP fusion, the fused MBP couldnot be cleaved by TEV digestion. XC6988 (glucose inhibited division protein B) tended to form gel-like precipitant in higher concentration, and showed no improvement under several different buffer conditions. XC1741 (reductase) was expressed as inclusion bodies under all tested conditions.
XC6774, an extracellular protease, was initially expressed as inclusion bodies in BL21 (DE3). It could be expressed using BL21 (DE3) pLys as the host cell, and when induction was preformed at 293K for 20 hour. However it has tendency to precipitate during purification. To solve this problem, further buffer screening (optimum solubility screen) was under-taken to find its optimum buffer condition. As a result, buffer of 20mM citric acid (pH=3.0) with 10% glycerol significantly increase XC6774 solubility even in high concentration. However, after screening in nearly 600 different conditions with purified protein, we were srill unable to obtain a suitable crystallization condition.
XC3857 was predicted as a YeeC-like protein. This protein family was much less studied before and no similar structure be found in the protein data bank (PDB). After expression and purification, XC3857 was similarly screened in nearly 600 conditions for crystallization but, again, we were unable to get any crystal. Since unsuitable buffer might cause protein to aggregate thus preventing crystal formation, we also used the optimum solubility screen method to find best buffer for XC3857. After testing 53 buffers, 20mM sodium phosphate (pH=6.5) was found to be the best solubility buffer, and crystals appeared in six different crystallization conditions. To improve crystal quality, the chosen conditions were further optimized manually and the crystallization plates were placed in a high-magnetic field.
XC296 was classified as an aldolase_II protein in the PFAM database. Compare to aldolase_I proteins, this class of aldolase was less studied; only one structure and its mutants from Escherichia coli are available in PDB. A ribulose epimerase with a class II aldolase-like fold from Escherichia coli has also been reported. Aldolase_II is important not only for its biological function, but also for its high stereo selectivity in enzymatic organic synthesis, which might be applicable in industry. Interestingly, XC296 has a shorter C-terminal end (less 20 a.a. residues) than fuculose 1-phosphate aldolase (aldolase II) from E. coli. Since the absence of fuculose 1-phosphate aldolase c-treminal rasidus has been proved to cause a drastic decrease in its activity and stereo selectivity, we consider that XC296 might have a similar, but different, mechnisum to reach its enzymatic activity and stereo selectivity. XC296 has now been successfully expressed and crystallized. Diffraction data have been collected and phases determined using the selenium multiple-wavelength anomalous dispersion (MAD) approach. The initial structure has been obtained and the model building and structural refinement are currently undergoing.
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