Improvement of the thermostability and catalytic activity of a mesophilic family 11 xylanase by N-terminus replacement
Section snippets
Materials
The P. pastoris expression kit including the pPIC9K vector, P. pastoris strain GS115, and Escherichia coli strain TOP10F′ were purchased from Invitrogen. Medium components were from Difco. Birchwood xylan was from Sigma Chemical. Restriction endonucleases were from TaKaRa. T4 DNA ligase and PCR kit were from Promega. Primers were synthesized by Sangon. The recombinant pBS-T anx plasmids containing the AnxA gene and pBS-T tfx plasmids containing TfxA gene were stored at −20 °C in our laboratory.
Construction of pPIC9k-atx plasmid and expression of ATx
Amino acid sequence comparison of catalytic domains of TfxA and AnxA is shown in Fig. 1. Fragments A, B, and atx were separated by electrophoresis and recovered (Fig. 2). It was confirmed that the nucleotide sequences of the amplified atx were 573 bp and identical with that of the theoretically designed by DNA sequencing. The nucleotide sequences of atx and its deduced amino acid sequences are shown in Fig. 3. Fig. 4 shows the schematic map of pPIC9K-atx plasmid.
The pPIC9K-atx plasmid was used
Discussion
As a eukaryote, P. pastoris is an attractive host for expression and production of heterologous protein for research and even for industrial purposes. The increasing popularity of this expression system can be attributed to several factors, most importantly: the techniques needed for the molecular genetic manipulation of P. pastoris are relatively simple; it can grow conveniently to high density levels in a simple and inexpensive medium; it is able to form a number of posttranslational
Acknowledgment
This work was supported by the National Natural Science Foundation of China (No. 30471254).
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