Identification, expression, and purification of a unique stable domain from human HSPC144 protein.
HSPC144 is a newly identified gene in human CD34(+) hematopoietic stem/progenitor cells. In this work, we have expressed and purified the 225-residue protein from Escherichia coli BL21 (DE3) and identified a stable fragment HSPC144-P (residues 44-225) by limited proteolysis method. The HSPC144-P fragment exhibits high stability with a little increase of secondary structure percentage as compared with the full-length protein. We anticipated that the N-terminally truncated protein possesses a more compact structure. By sequence analysis, the proteolytic fragment shares a great similarity with DUF589 domain, a previously identified domain with unknown function. This novel domain is highly conserved in Thy28 proteins and is worthy of structural and functional studies. We have subcloned this homologous domain from HSPC144 protein and purified to homogeneity for structure analysis. The (15)N and (15)N/(13)C-labeled DUF589 domain samples have been prepared successfully and determination of the NMR structure is in progress.
Song
Ai-Xin
AX
Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.
Chang
Yong-Gang
YG
Gao
Yong-Guang
YG
Lin
Xiao-Jing
XJ
Shi
Yan-Hong
YH
Lin
Dong-Hai
DH
Hang
Qiu-Hua
QH
Hu
Hong-Yu
HY
D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002942:Circular Dichroism; D019286:Endopeptidase K / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019906:Nuclear Magnetic Resonance, Biomolecular; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization
D009687:Nuclear Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C505706:THYN1 protein, human; D019286:Endopeptidase K