Construction and characterization of different MutS fusion proteins as recognition elements of DNA chip for detection of DNA mutations

摘要

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-His(6)-GFP-(Ser-Gly)(6)-MutS (THGLM), Trx-His(6)-(Ser-GlY)(6)-Strep tagII-(Ser-GlY)(6)-MutS (THLSLM) and Trx-His(6)-(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 similar to 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 Fluorolink (TM) 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. (c) 2004 Elsevier B.V. All rights reserved.