Biomedical subjects
Publications and source records attributed to J S Huston.
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The functional antigen binding region of antidinitrophenol mouse IgA myeloma MOPC 315 has been produced as a single-chain Fv (sFv) protein in E. coli. Recombinant 315 proteins included sFv alone, a bifunctional fusion protein with amino-terminal fragment B (FB) of staphylococcal protein A, and a two-chain 315 Fv fragment. Successful refolding of the 315 sFv required formation of disulfide bonds while the polypeptide was in a denatured state, as previously observed for the parent Fv fragment. Affinity-purified recombinant 315 proteins showed full recovery of specific activity, with values for Ka,app of 1.5 to 2.2 x 10(6) M-1, equivalent to the parent 315 Fv fragment. As observed for natural 315 Fv, the sFv region of active FB-sFv315 fusion protein was resistant to pepsin treatment, whereas inactive protein was readily degraded. These experiments will allow the application of protein engineering to the 315 single-chain Fv; such studies can advance structure-function studies of antibody combining sites and lead to an improved understanding of single-chain Fv proteins.
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A bifunctional molecule was genetically engineered which contained an amino-terminal effector domain that bound immunoglobulin Fc (fragment B of staphylococcal protein A) and a carboxyl-terminal domain that bound digoxin [a single-chain Fv (sFv)]. Effector and sFv binding properties were virtually identical with those of the parent molecules, despite the proximity of the FB to the sFv combining site. This finding is unprecedented since in all molecules of the natural immunoglobulin superfamily, the antigen binding domain is amino terminal to the effector domain. The FB-sFv sequence was encoded in a single synthetic gene and expressed as a 33,106 molecular weight protein in Escherichia coli. After purification, renaturation, and affinity isolation, yield of active fusion protein were 110 mg/L of fermented cells (18.5-g cell paste). Bifunctionality was confirmed by the ability of FB-sFv to cross-link IgG to digoxin-bovine serum albumin, as measured by plate assays and by Ouchterlony analysis. Analysis of the expressed fusion protein suggests that the sFv holds promise for the development of multifunctional, targetable single-chain proteins.
Mouse epidermal growth factor (mEGF) and the beta subunit of follicle-stimulating hormone (hFSH) (hFSH-beta) have been shown to inhibit binding of intact hFSH to its testes membrane receptor in vitro. Both hFSH-beta and mEGF contain the tetrapeptide sequence Thr-Arg-Asp-Leu (TRDL). Previous results demonstrated that synthetic TRDL inhibited binding of intact hFSH to receptor. We therefore investigated the possibility that TRDL was located on an exposed region of FSH-beta using a polyclonal antiserum to hFSH [NHPP anti-hFSH batch 4 (AB4)] which recognized determinants on intact hFSH and its beta subunit, but not the alpha subunit. Pituitary FSH preparations from several mammalian species produced parallel inhibition curves in a heterologous [AB4 and 125I-labeled ovine FSH (125I-oFSH)] radioimmunoassay with relative potencies similar to those observed for the same preparations assayed by radioligand receptor assay. This antiserum also competitively inhibited 125I-FSH binding to receptor. Thus, AB4 appeared to recognize antigenic determinants that are highly conserved and located at or near regions involved with hormone recognition of receptor for FSH. Synthetic TRDL inhibited 50% of 125I-hFSH binding to antiserum at a concentration of 1.36 mg/tube (9 x 10(-3) M). Other tetrapeptides (Thr-Pro-Arg-Lys and Lys-Thr-Cys-Thr) had no inhibitory activity at comparable concentrations. A mixture of the free amino acids T, R, D, and L inhibited radioligand binding only at significantly higher concentrations than TRDL.(ABSTRACT TRUNCATED AT 250 WORDS)
A biosynthetic antibody binding site, which incorporated the variable domains of anti-digoxin monoclonal antibody 26-10 in a single polypeptide chain (Mr = 26,354), was produced in Escherichia coli by protein engineering. This variable region fragment (Fv) analogue comprised the 26-10 heavy- and light-chain variable regions (VH and VL) connected by a 15-amino acid linker to form a single-chain Fv (sFv). The sFv was designed as a prolyl-VH-(linker)-VL sequence of 248 amino acids. A 744-base-pair DNA sequence corresponding to this sFv protein was derived by using an E. coli codon preference, and the sFv gene was assembled starting from synthetic oligonucleotides. The sFv polypeptide was expressed as a fusion protein in E. coli, using a leader derived from the trp LE sequence. The sFv protein was obtained by acid cleavage of the unique Asp-Pro peptide bond engineered at the junction of leader and sFv in the fusion protein [(leader)-Asp-Pro-VH-(linker)-VL]. After isolation and renaturation, folded sFv displayed specificity for digoxin and related cardiac glycosides similar to that of natural 26-10 Fab fragments. Binding between affinity-purified sFv and digoxin exhibited an association constant [Ka = (3.2 +/- 0.9) x 10(7) M-1] that was about a factor of 6 smaller than that found for 26-10 Fab fragments [Ka = (1.9 +/- 0.2) x 10(8) M-1] under the same buffer conditions, consisting of 0.01 M sodium acetate, pH 5.5/0.25 M urea.
Two tetrapeptide sequence homologies between mouse epidermal growth factor precursor (mEGFP) and human follitropin (FSH) were revealed by a computer program that identifies identical residues among polypeptide sequences. The two tetrapeptides, Lys-Thr-Cys-Thr (KTCT) and Thr-Arg-Asp-Leu (TRDL), are present in the hormone-specific beta subunit of FSH from all species studied. These tetrapeptides are not present in the alpha subunit, which is common to all pituitary glycoprotein hormones. Both tetrapeptides are also found in mEGFP, and one tetrapeptide, TRDL, is located within the 53-residue form of mEGF purified from mouse submaxillary glands. Computer-generated hydropathy profiles predicted that both tetrapeptides are located in hydrophilic portions of the FSH beta subunit and that TRDL is in a hydrophilic portion of commercially available mEGF. Therefore, the tetrapeptides might be accessible to receptor binding sites for FSH. We report that mEGF inhibits binding of 125I-labeled human FSH to receptors in testis by 50% (I50) at a concentration of 1.8 X 10(-5) M. No binding inhibition was observed by GnRH or arginine-vasopressin at 10(-4) M, neither of which contain the tetrapeptide sequences. FSH beta subunit, which contains both tetrapeptides, also inhibited binding (I50 = 9 X 10(-8) M) of 125I-labeled human FSH to testis receptor. Thus, it appears that FSH beta subunit and mEGF are capable of inhibiting binding of FSH to testicular FSH receptors, presumably through interactions that include the homologous tetrapeptides. This presumption was supported by the observation that the synthetic tetrapeptides (KTCT or TRDL) were also active in inhibiting binding of 125I-labeled human FSH to testis receptor.
In this study, polyclonal antisera to human FSH (hFSH) and its alpha- and beta-subunits have been used as probes of the interaction between hFSH and the FSH receptor from bovine testis. Preincubation of [125I]iodo-hFSH with antisera to the subunits of hFSH induced an augmentation in binding of radioiodinated hFSH to membrane receptors. If the antisera were made monovalent by papain digestion, all binding augmentation previously seen with bivalent antisera was eliminated. Furthermore, whereas bivalent antiserum to intact hFSH had no effect on [125I]iodo-hFSH binding to receptor, papain-generated monovalent antisera to hFSH strongly inhibited [125I]iodo-hFSH binding. Formation of [125I]iodo-hFSH-receptor complex reduces the ability of bivalent antibodies to FSH or its subunits to interact with the complex, as determined by immunoprecipitation studies. Our studies suggest that each FSH subunit interacts with the membrane receptor. These results also caution that a comparison of bivalent and monovalent antisera is necessary to determine if a particular antibody effects binding of hormone to receptor.
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Glial fibrillary acidic protein was purified from 4 M urea extracts of bovine brain by DEAE Bio-Gel A chromatography, 30% ammonium sulfate precipitation and hydroxylapatite chromatography. Subunits of about 54 000 daltons are present in solution as polydisperse distributions of polymers largely constrained by the presence of interchain disulfide linkages. Circular dichroism measurements indicate a native conformation containing some alpha-helical structure. The relevance of these findings to the cytoskeletal function of intermediate (80-100 A) filaments is discussed.
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The single-chain Fv (sFv) has proven attractive for immuno-targeting, both alone and as a targeting element within sFv fusion proteins. This chapter summarizes the features of sFv proteins that have sparked this interest, starting with the conservation of Fv architecture that makes general sFv design practical. The length and composition of linkers used to bridge V domains are discussed based on the sFv literature; special emphasis is given to the (Gly4Ser)3 15-residue linker that has proven of broad utility for constructing Fv regions of antibodies and other members of the immunoglobulin superfamily. The refolding properties of sFv proteins are summarized and examples given from our laboratory. Spontaneous refolding from the fully reduced and denatured state, typified by 26-10 sFv, is contrasted with disulfide-restricted refolding, exemplified by MOPC 315 and R11D10 sFv proteins, which recover antigen binding only if their disulfides have been oxidized prior to removal of denaturant. The medical value of sFv proteins hinges on their reliability in antigen recognition and rapidity in targeted delivery. Detailed analysis of specificity and affinity of antigen binding by the 26-10 antidigoxin sFv has demonstrated very high fidelity to the binding properties of the parent 26-10 sFv. These results gave confidence to the pursuit of more complex biomedical applications of these proteins, which is indicated by our work with the R11D10 sFv for the imaging of myocardial infarctions. Diagnostic imaging and therapeutic immunotargeting by sFv present significant opportunities, particularly as a result of their pharmacokinetic properties. Intravenously administered sFv offers much faster clearance than conventional Fab fragments or intact immunoglobulin with minimal background binding.