Aim. The obtaining of bioaffinity sorbent based on the immobilized protein A of S. aureus (SPA) using two cellulose-binding domains (CBD), and its application for purification of antibodies. Methods. The DNA sequences encoding SPA and two CBD were genetically fused, expressed in the high-productive Escherichia coli system and the protein SPA-CBD2 was obtained in a soluble form. The SPA-CBD2 fusion protein was affinity immobilized on the microcrystalline cellulose. Results. Capacity of bioaffinity sorbent (1 mg SPA-CBD2/1 ml CC31-cellulose), dynamic capacity (3 mg mouse IgG/1 ml bioaffinity sorbent), efficiency and stability during prolonged storage were determined. The bioffinity sorbent was used for purification of antibodies. The purity of antibodies in eluted fractions was more than 95 %. The purified antibodies detected target antigens with a high sensitivity. Conclusions. The designed bioaffinity sorbent provides obtaining pure poly- and monoclonal antibodies in functionally active form and can be useful for the fractionation of mouse immunoglobulin G
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
A combinatorial cDNA library of mouse variable immunoglobulin genes from mice immunized with recombinant human interferon β 1b (rhIFN-β 1b) has been constructed. For this purpose, cDNA of variable genes of heavy ( V H ) and light ( V L ) immunoglobulin chains amplified from splenocytes were joined by linker DNA to form single-chain antibodies (single-chain Fv-antibodies, or ScFv's). The ScFv-DNA pool thus obtained was cloned into a phagemid vector and used to transform Escherichia coli. Bacterial clones that produce single-chain antibodies that are specific to rhIFN-β 1b ScFv's were selected using the technique of phage display. Such characteristics of generated library as abundance, functional size, and initial diversity of the ScFv-DNA sequences were established in the study. A high degree of specificity of interaction of selected phage displayed ScFv's with rhIFN-β 1b has been demonstrated.
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