A sulfoglycolipid beta-sulfoquinovosyldiacylglycerol (beta SQDG) binds to Met1-Arg95 region of murine DNA polymerase lambda (Mmpol lambda) and inhibits its nuclear transit

作者：Takakusagi K; Takakusagi Y; Ohta K; Aoki S; Sugawara F; Sakaguchi K^*

来源：Protein Engineering Design and Selection, 2010, 23(2): 51-60.

DOI：10.1093/protein/gzp064

• DNA polymerase lambda
• nucleartransit
• sulfoquinovosylacylglycerol
• T7 phage display

Beta-sulfoquinovosyldiacylglycerol (beta SQDG) is a synthetic sulfoglycolipid that shows inhibitory activity of DNA polymerase lambda (pol lambda). Here we identified a beta SQDG binding region within murine pol lambda (Mmpol lambda) using T7 phage display technology. We compared the binding intensity of bSQDG with recombinant phages (phages lambda 1-6) that displayed different segments of Mmpol lambda. The binding assay clearly showed that phage lambda 1, which displayed the non-structural Met1-Arg95 region including the nuclear localization signal (NLS) and part of the BRCT domain, bound more strongly to beta SQDG than the other recombinant phages. Binding assays using recombinant proteins gave similar results, showing specific beta SQDG binding to Met1-Arg95 with a K(D) value of 9.9 nM. Furthermore, in a cell-based assay, nuclear localization of EGFP-pol lambda was inhibited in the presence of beta SQDG possibly due to binding of beta SQDG to NLS. These experiments clearly show that the binding region of beta SQDG within Mmpol l could be successfully identified using T7 phage display technology. We suggest that the strategy we describe here will be of value for identifying the binding site within a protein for small ligands, and will provide information that cannot be obtained using other experimental techniques due to their inherent technical limitations.

• 出版日期2010-2