[Cloning and expression of xyn III from genomic DNA of Trichoderma reesei QM9414 by overlap-PCR].
After the cell enters into its programmed cell death, xylanases from grass plants gradually matured through its N-terminal and C-terminal sequence been cut by acid proteases several times. They could not be expressed by conventional protein expression system. Search the GenBank database, xynIII from a mutant of T. reesei QM9414(ATCC26921)was found. It is similar to grass plants' xylanase in their families and structures. It couldn't express in T. reesei QM9414, but its gene exist in genomic DNA as one copy. Through overlap-PCR method, 4 exons of xynIII were cloned, sequenced, spliced, and the whole cDNA of mature xynIII was acquired. The cDNA was inserted into pETBlue-2 vector and transformed into E. coli DE3 pLacI cell. Xyn III could be expressed in the transformed cell under the conditions of 37 degrees C, 1 mmol/L IPTG induced for 3h. Low temperature (15 degrees C), long time(64h) induction(0.2 mmol/L IPTG) could enhance xynIII activity.
The Key Laboratory of Bioresources Technology of Jiangsu Province, The Key Laboratory of Microbial Technology of Nanjing Normal University, College of Life Science, Nanjing Normal University, Nanjing 210097, China.
Chang-Mei
CM
Lu
Sheng
S
Yuan
Qing-Xin
QX
Zhao
D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D043364:Endo-1,4-beta Xylanases / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D014242:Trichoderma / Q000235:genetics*
D018076:DNA, Complementary; D043364:Endo-1,4-beta Xylanases