Immobilized single chain antibodies for affinity purification of recombinant human IFN-α2b

Abstract

Im mo bi lized sin gle chain an ti bod ies for af fin ity pu ri fi ca tion of re com bi nant hu man ifn a2b P.V. Gilchuk, O.V. Okunev, D.M. Irodov, M.V. Pav lo va, O.Ya. Yakovenko The In sti tute of Mo lec u lar Bi ol ogy and Ge net ics, NAS of Ukraine 150, Ac a de mi cian Zabolotny Str., Kyiv, 03143, Ukraine 1 Na tional Taras Shevchenko Uni ver sity of Kyiv 64, Volodymyrska Str., Kyiv, 01033, Ukraine gilchuk@ukr.netA lab o ra tory method for ob tain ing immunoaffinity me dia for chro mato graphic pu ri fi ca tion of pro teins based on ori ent ing and non co va lent im mo bi li za tion of re com bi nant an ti body frag ments on cel lu lose ma trix is de scribed. The sin gle chain an ti body (ScFv) against hu man IFN a2b was ge net i cally fused to cel lu lose bind ing do main (CBD) from Clostridium thermocellum cellulosome and ex pressed in Esch e richia coli. Af ter the iso la tion of the tar get pro tein in func tion ally ac tive form from bac te ria cells the di rected im mo bi li za tion on microgranular cel lu lose has been car ried out. The cel lu lose with im mo bi lized ScFv CBD fu sion was used as af fin ity me dia to per form the pu ri fi ca tion of re com bi nant hu man IFN a2b. Keywords: sin gle chain an ti bod ies, immunoaffinity chro ma tog ra phy, pro tein im mo bi li za tion, fu sion pro tein. 157 ISSN 0233 7657. Biopolymers and cell. 2006. Vol. 22. ISS 2. Translated from Ukrainia. ã P.V. Gilchuk, O.V. Okunev, D.M. Irodov, M.V. Pav lo va, O.Ya. Yakovenko

Development of the chromatographic medium for the affinity isolation of the recombinant hIFN-β1b based on immobilized single-chain antibodies

“…As a spacer for the spatial separation of two affi nity centers of the chimeric protein (CBD and ScFv) a sequence of 13 amino acids was introduced [5,6]. Professor Y. Shoham (Israel) kindly provided the plasmid vector pCBD with the cellulose binding domain (CBD) sequence from Clostridium thermocellum for our research.…”

“…Refolding of the purifi ed ScFv bINF -CBD was performed by stepwise dilution [6]. After renaturation ScFv bINF -CBD was immobilized on chitin beads (New England Biolabs, UK).…”

40 years within two epochs of two millennia

“…There are no separate parts in the organism, everything interacts, inter-coordinates, interchanges, etc. In this area, the department was elaborating the cytokine therapy (restoration of blood supply of the ischemic kidney) [42][43], gene therapy (simulations of diabetes and atherosclerosis) [22][23][24][25][26][27][28][29], cell therapy (on the basis of the protocol of obtaining mesenchymal multipotent cells from gelatin of Wharton) [44]. As for the precise technological support (purification, testing, delivery, etc.…”

The development of strategy for obtaining single-chain recombinant antibodies against cell-surface biomarkers on the example of human CD34

“…Ре ком би нан тный антиген rhExCD34 очищали и ре на ту рировали из те лец вклю че ния ме тодом им мо би ли за ци он ной ме талл-аф фин ной хро матог ра фии (IMAX) [7].…”