Expression, purification, crystallization and preliminary X-ray crystallographic analysis of Enpp1

Abstract

Enpp1 is an extracellular membrane-bound glycoprotein that regulates bone mineralization by hydrolyzing ATP to generate pyrophosphate. The extracellular region of mouse Enpp1 was expressed in HEK293S GnT1 À cells, purified using the TARGET tag/P20.1-Sepharose system and crystallized. An X-ray diffraction data set was collected to 3.0 Å resolution. The crystal belonged to space group P3 1 , with unit-cell parameters a = b = 105.3, c = 173.7 Å . A singlewavelength anomalous dispersion (SAD) data set was also collected to 2.7 Å resolution using a selenomethionine-labelled crystal. The experimental phases determined by the SAD method produced an interpretable electron-density map.

ENPP1 processes protein ADP‐ribosylation in vitro

“…Of note, HEK293S GnT1 − were used to express mENPP2‐1‐T, as described in Kato et al . . To produce mENPP2‐1‐8xHis (ENPP1 – OPPF 17442), the full‐length gene for ENPP2‐1 containing the native signal sequence (bp 1–2619) was amplified by PCR using Phusion Flash polymerase (Life Technologies, Thermo Fisher Scientific, Hemel Hempstead, UK) and the following primers containing the extensions necessary for ligation‐independent cloning (Forward primer:aggagatataccatgATGGCAAGACAAGGCTGTTTCGG and reverse primer:gtgatggtgatgtttGTCTTCTTGGCTGAAGATTGGCAAATGT).…”

“…pGEX-4T1 GST-PARP10cd (amino acids 818-1025) plasmid was a gift from Bernhard L€ uscher (RWTH Aachen University) [17]. GST [48,49]. In particular, the extracellular region of mouse Enpp1 (residues 92-905) was fused with the secretory signal sequence (residues 1-50) and the N-terminal nine residues of the somatomedin B-like 1 (SMB1) domain (residues 51-59) of mouse Enpp2 at the N terminus and with the TARGET tag at the C terminus to generate a secreted ENNP2-1 chimera (mENPP2-1-T) [48,49].…”

“…GST [48,49]. In particular, the extracellular region of mouse Enpp1 (residues 92-905) was fused with the secretory signal sequence (residues 1-50) and the N-terminal nine residues of the somatomedin B-like 1 (SMB1) domain (residues 51-59) of mouse Enpp2 at the N terminus and with the TARGET tag at the C terminus to generate a secreted ENNP2-1 chimera (mENPP2-1-T) [48,49]. Of note, HEK293S GnT1 À were used to express mENPP2-1-T, as described in Kato et al [48,49].…”

“…In particular, the extracellular region of mouse Enpp1 (residues 92-905) was fused with the secretory signal sequence (residues 1-50) and the N-terminal nine residues of the somatomedin B-like 1 (SMB1) domain (residues 51-59) of mouse Enpp2 at the N terminus and with the TARGET tag at the C terminus to generate a secreted ENNP2-1 chimera (mENPP2-1-T) [48,49]. Of note, HEK293S GnT1 À were used to express mENPP2-1-T, as described in Kato et al [48,49]. To produce mENPP2-1-8xHis (ENPP1 -OPPF 17442), the full-length gene for ENPP2-1 containing the native signal sequence (bp 1-2619) was amplified by PCR using Phusion Flash polymerase (Life Technologies, Thermo Fisher Scientific, Hemel Hempstead, UK) and the following primers containing the extensions necessary for ligation-independent cloning (Forward primer: aggagatataccatgATGGCAAGACAAGGCTGTTTCGG and reverse primer:gtgatggtgatgtttGTCTTCTTGGCTGAAGAT TGGCAAATGT).…”

Crystal structure of Enpp1, an extracellular glycoprotein involved in bone mineralization and insulin signaling

“…The extracellular region (residues 92-905) of mouse Enpp1 was expressed, purified, and crystallized as described previously (36). X-ray diffraction data were collected at 100 K on beamline BL32XU at SPring-8 (Hyogo, Japan).…”