Construction and characterization of different MutS fusion proteins as recognition elements of DNA chip for detection of DNA mutations.
Three MutS fusion systems were designed as the mutation recognition and signal elements of DNA chips for detection of DNA mutations. The expression vectors containing the encoding sequences of three recombinant proteins, Trx-His6-GFP-(Ser-Gly)6-MutS (THGLM), Trx-His6-(Ser-Gly)6-Strep tagII-(Ser-Gly)6-MutS (THLSLM) and Trx-His6-(Ser-Gly)6-MutS (THLM), were constructed by gene slicing in vitro. THGLM, THLSLM and THLM were then expressed in Escherichia coli AD494(DE3), respectively. SDS-PAGE analysis revealed that each of the expected proteins was approximately 30% of the total bacterial proteins. The recombinant proteins were purified to the purity over 90% by immobilized metal (Co2+) chelation affinity chromatography. Bioactivity assay indicated that three fusion proteins retained the mismatch-binding activity and the functions of other fusion partners. DNA chips arrayed both mismatched and unpaired DNA oligonucleotides as well as rpoB gene from Mycobacterium tuberculosis were prepared. THGLM, THLSLM and THLM that was labeled with Fluorolinktrade mark Cy3 reactive dye, were then used as both mutation recognition and labeling elements of DNA chips. The resulting DNA chips were used to detect the mismatched and unpaired mutations in the synthesized oligonucleotides and single base mutation in rpoB gene of M. tuberculosis that is resistant to rifamycin.
Bi
Li-Jun
LJ
Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101, People's Republic of China.
Zhou
Ya-Feng
YF
Zhang
Xian-En
XE
Deng
Jiao-Yu
JY
Wen
Ji-Kai
JK
Zhang
Zhi-Ping
ZP
D002232:Carbocyanines; D004252:DNA Mutational Analysis / Q000379:methods*; D004269:DNA, Bacterial / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D051719:MutS DNA Mismatch-Binding Protein / Q000138:chemical synthesis / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000235:genetics* / Q000378:metabolism
D002232:Carbocyanines; D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; C089187:cyanine dye 3; D051719:MutS DNA Mismatch-Binding Protein