Overexpression and purification of single zinc finger peptides of human zinc finger protein ZNF191.
ZNF191, a new human zinc finger protein, probably relates to some hereditary diseases and cancers. To obtain structural information of zinc finger domain a convenient method for obtaining milligram quantities of each zinc finger peptide of ZNF191 is necessary. Here, we report an Escherichia coli expression system for rapid and high-level expression of zinc finger 3 and zinc finger 4 of ZNF191. The gene of zinc finger 3 or zinc finger 4 was cloned into pET31b vector to allow expression of single zinc finger peptide as a ketosteroid isomerase (KSI) fusion protein. The KSI-single zinc finger fusion protein was overexpressed in the form of inclusion body, which can be purified by washing several times using buffer solutions, and then be cleaved directly by cyanogen bromide to release single zinc finger peptide. The more than 20mg/L yield of single zinc finger peptide was achieved with more than 95% purity by using YM ultrafiltration membranes. Circular dichroism spectra of these two single zinc finger peptides titrated with Zn(2+) ions demonstrate that they have different secondary structures.
Zhao
Dong-Xin
DX
Chemical Biology Lab, Department of Chemistry, Fudan University, Shanghai 200433, PR China.
Ding
Zhi-Chun
ZC
Liu
Yu-Qi
YQ
Huang
Zhong-Xian
ZX
D002021:Buffers; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003488:Cyanogen Bromide / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005561:Formates / Q000494:pharmacology; D005612:Freeze Drying; D005822:Genetic Vectors; D006639:Histidine / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D002479:Inclusion Bodies / Q000378:metabolism; D051741:Kruppel-Like Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008567:Membranes, Artificial; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D010957:Plasmids; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D012996:Solutions / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D014462:Ultrafiltration; D015032:Zinc / Q000737:chemistry; D016335:Zinc Fingers
D002021:Buffers; D005561:Formates; D051741:Kruppel-Like Transcription Factors; D008567:Membranes, Artificial; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D012996:Solutions; C457300:ZNF24 protein, human; C030544:formic acid; D006639:Histidine; D015032:Zinc; D003488:Cyanogen Bromide