[Overproduction of single-chain antibodies against human IFN-alpha2B in Escherichia coli and their isolation in biologically active form].
The gene encoding mouse single chain antibody (ScFv) against human interferon alpha2b (IFN-alpha2b) was cloned into the plasmid vector under the control of promoter from phage T7 and the recombinant protein was expressed in Escherichia coli as inclusion bodies. After the isolation of inclusion bodies the desired protein containing affinity tail "6His tag" was solubilized and purified under denaturing conditions by immobilized-metal affinity chromatography. The soluble and purified ScFv was obtained by "on column" refolding and the recovery of biological activity were demonstrated. The higher levels of ScFv production for intracellular expression system in comparison with ScFv obtained by secretion were shown. The advantages of described refolding method are simplicity and high efficacy, moreover, refolding using a chromatographic process represents the manufacturable approach because it is easily automated using commercially available materials and preparative chromatography systems and also can be combined with simultaneous purification.
Hil'chuk
P V
PV
Okuniev
O V
OV
Irodov
D M
DM
Pavlova
M V
MV
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000235:genetics; D002479:Inclusion Bodies; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000276:immunology*; D051379:Mice; D010957:Plasmids; D020673:Protein Renaturation; D011994:Recombinant Proteins
D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011994:Recombinant Proteins; C055966:immunoglobulin Fv