Protein complex purification from Thermoplasma acidophilum using a phage display library

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• 作者：Á ; gnes Hubert^a ; ^{hubert@biochem.mpg.de" class="auth_mail} ; Yasuo Mitani^b ; ^{mitani-y@aist.go.jp" class="auth_mail} ; Tomohiro Tamura^c ; ^{t-tamura@aist.go.jp" class="auth_mail} ; Marius Boicu^a ; ^{boicu@biochem.mpg.de" class="auth_mail} ; Istvá ; n Nagy^a ; ^{nagy@biochem.mpg.de" class="auth_mail}
• 关键词：EM ; electron microscopy ; IMAC ; immobilized metal affinity chromatography ; MS/MS ; tandem mass spectrometry ; Ni&ndash ; NTA ; nickel&ndash ; nitriloacetic acid ; OHA-300 ; 300  ; mM K2HPO4&ndash ; KH2PO4 elution fraction of Sup12-HMWF ; OHA-500 ; 500  ; mM K2HPO4&ndash ; KH2PO4 elution fraction of Sup12-HMWF ; scFv ; single-chain variable fragment ; SEC ; size exclusion chromatography ; Sup12-HMWF ; Superose12-separated high molecular weight protein fraction of T. acidophilum cytosolic extract ; TAP ; tandem affin
• 刊名：Journal of Microbiological Methods
• 出版年：March, 2014
• 年：2014
• 卷：98
• 期：Complete
• 页码：15-22
• 全文大小：1154 K

文摘

We developed a novel protein complex isolation method using a single-chain variable fragment (scFv) based phage display library in a two-step purification procedure. We adapted the antibody-based phage display technology which has been developed for single target proteins to a protein mixture containing about 300 proteins, mostly subunits of Thermoplasma acidophilum complexes. T. acidophilum protein specific phages were selected and corresponding scFvs were expressed in Escherichia coli. E. coli cell lysate containing the expressed His-tagged scFv specific against one antigen protein and T. acidophilum crude cell lysate containing intact target protein complexes were mixed, incubated and subjected to protein purification using affinity and size exclusion chromatography steps. This method was confirmed to isolate intact particles of thermosome and proteasome suitable for electron microscopy analysis and provides a novel protein complex isolation strategy applicable to organisms where no genetic tools are available.