Abstract
Expression of modified xynA gene fragments in Escherichia coli BL21 was studied, using the complete xynA gene from Bacillus subtilis BE-91 as the positive control. The technical workflow consisted of the following steps: (1) predicting protein structures relative to the xynA gene; (2) designing primers for modifiers; (3) amplifying the modifiers; (4) integrating the modifiers with the pET-28a(+) vector; (5) transferring the recombinant plasmids into E. coli BL21; (6) evaluating and analyzing the expression of modified cells. The results were: (1) the xynA gene from BE-91 with the untranslated region deleted on both ends was able to promote XynA activity by 28.9 %; (2) deletion of the 1- to 16-amino acid (AA) coding sequence in the open reading frame on the 5′-end, deletion of the 209- to 213-AA fragment on the 3′-end and deletion of the 20 AA on both ends could promote XynA activity by 27.2, 27.7 and 24.0 %,respectively; (3) deletion of the 1- to 29-AA fragment on the 5′-end and deletion of the 197- to 213-AA fragment on the 3′-end could reduce XynA activity dramatically by 95.6 and 74.8 %, respectively; (4) inactivation factors of XynA would be either the first β-fold and the hydrophilic structure domain or the last two α-screws and the seventeenth turn region. The results mean that any deletion in the catalytic domain would lead to a decline or inactivation in XynA activity while the deletion of any sequence outside the catalytic domain could effectively promote XynA activity, as such sequences are unnecessary for XynA function.
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The present work was supported by a grant from the National Project for High-technology Research (2006AA02Z249) and also funded by China Agriculture Research System (CARS-19-E24).
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Z. Liu and J. Xu contributed equally to this work
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Liu, Z., Xu, J., Duan, S. et al. Expression of modified xynA gene fragments from Bacillus subtilis BE-91. Ann Microbiol 64, 139–145 (2014). https://doi.org/10.1007/s13213-013-0642-9
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DOI: https://doi.org/10.1007/s13213-013-0642-9