Abstract
Single-chain Fv (sFv) proteins are genetically engineered molecules that consist of the two variable domains of an antibody connected by a polypeptide linker; they contain the antigen binding function of the parental protein in a single 30-kDa polypeptide chain. sFvs are usually produced in bacteria where they are insoluble and therefore require extensive refolding in vitro. In this report we followed the processing of three antibody sFvs (145-2C11 directed against murine CD3 epsilon chain, OKT9 against the human transferrin receptor, and U7.6 against dinitrophenyl groups) by transfected mammalian (COS-7) cells to determine whether the mammalian protein folding machinery can produce and secrete active sFv with high efficiency. The sFvs contained an immunoglobulin light chain leader sequence, which directed them to the endoplasmic reticulum and allowed secretion into the medium. We found that the sFvs were secreted at different rates, with the rate-limiting step of secretion being their exit from the endoplasmic reticulum. We increased the secretion rate of one of the sFvs by introducing an asparagine-linked glycosylation site in FR1 of the heavy chain, and by using tunicamycin (an inhibitor of glycosylation) we found that glycosylated antibody sFvs were secreted faster than their nonglycosylated counterparts. All secreted sFvs specifically bound their antigens; where tested, at least 90% of the secreted sFv was functional. Therefore, mammalian cells can effectively fold and secrete sFv antibody and can provide a convenient system for testing and producing sFv proteins.
Highlights
Introduction of n GlyosvlntionSitr in thrU7.6-sFll Enhnnccs Its Sccrrtion Xntc-Of the antibody Single-chain Fv (sFv) constructs, theU7.6-sFv was secreted much more slowly than the others (Fig.CDR3 of VI, (-Cys in Fig. 4).and second, we introduced an asparagine-linked glycosylation site in the same positiaosnthe glycosylation site in OKTS ( + A m ) .A third construct incorporated both mutations (-Cys+Asn)
A different approach to produce active sFv would be t o use the more sophisticated refolding machinery that is located in the endoplasmic reticulum (ER) of mammalian cells
Site-directed Mutagenesis of U7.6-Mutations in the U7.6-sFv constructs were introduced using a TYansformer Mutagenesis Kit (Clonetech Laboratories Inc., Palo Alto, CA) according to the manufacturer's samples gave standard deviations in band densities of less than 10%. sFu Binding Studies-The specificity of sFv binding was tested using the radiolabeled material present in the media of transfected COS-7 cells after either a 2-h or a 6-h chase
Summary
Vol 269, No 42, Issue of October 21, pp. 26267-26273, 1994 Printed in U.S.A. Carolina R. SFvs are usually produced in bacteria where they are insoluble and require extensive refolding in uitro In this reportwe followedthe processing of three antibody sFvs (145-X11 directed against murine CD3 E chain, OKT9 against the human transferrin receptor, and U7.6 against dinitrophenyl groups) by transfected mammalian (COS-7) cells to determine whether the mammalian protein folding machinery can produce and secrete active sFv with high efficiency. A different approach to produce active sFv would be t o use the more sophisticated refolding machinery that is located in the endoplasmic reticulum (ER) of mammalian cells The potential benefitof this approach could be substantial, since the ER contains enzymes that catalyze specific isomerization steps, buitt contains a number of chaperones that aid in the folding process and prevent thesecretion of incorrectly. The abbreviations used are: Fv, the portion of immunoglobulin molecules consisting of the light and heavychain variabledomains; V variable; H, heavy; L, light; DNP, 2,4-dinitrophenyl; sFv, single-chain
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