Parallel cloning, expression, purification and crystallization of human proteins for structural genomics

54 human genes were selected as test targets for parallel cloning, expression, purification and crystallization. Proteins from these genes were selected to have a molecular weight of between 14 and 50 kDa, not to have a high percentage of hydrophobic residues (i.e. more likely to be soluble) and to have no known crystal structures and were not known to be subunits of heterocomplexes. Four proteins containing transmembrane regions were selected for comparative tests. To date, 44 expression clones have been constructed with the Gateway(TM) cloning system (Invitrogen, The Netherlands). Of these, 35 clones were expressed as recombinant proteins in Escherichia coli strain BL21 (DE3)-pLysS, of which 12 were soluble and four have been purified to homogeneity. Crystallization conditions were screened for the purified proteins in 96-well plates under oil. After further refinement with the same device or by the hanging-drop method, crystals were grown, with needle, plate and prism shapes. A 2.12 Angstrom data set was collected for protein NCC27. The results provide insights into the high-throughput target selection, cloning, expression and crystallization of human genomic proteins.

Ren, H (författare)
Chen, Q (författare)
Fang, G (författare)
Li, LF (författare)
Li, R (författare)
Wang, R (författare)
Jia, XY (författare)
Liang, YH (författare)
Hu, MH (författare)
Li, Y (författare)
Luo, JC (författare)
Gu, XC (författare)
Su, Xiao-DongLund University,Lunds universitet,Biokemi och Strukturbiologi,Centrum för Molekylär Proteinvetenskap,Kemiska institutionen,Institutioner vid LTH,Lunds Tekniska Högskola,Biochemistry and Structural Biology,Center for Molecular Protein Science,Department of Chemistry,Departments at LTH,Faculty of Engineering, LTH(Swepub:lu)mbfys-xs (författare)
Luo, M (författare)
Lu, SY (författare)
Biokemi och StrukturbiologiCentrum för Molekylär Proteinvetenskap (creator_code:org_t)

Ingår i:Acta Crystallographica. Section D: Biological Crystallography58, s. 2102-21081399-0047

Av författaren/redakt...: Ding, HT; Ren, H; Chen, Q; Fang, G; Li, LF; Li, R; visa fler...; Wang, R; Jia, XY; Liang, YH; Hu, MH; Li, Y; Luo, JC; Gu, XC; Su, Xiao-Dong; Luo, M; Lu, SY; visa färre...

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