Mammalian expression and hollow fiber bioreactor production of recombinant anti-CEA diabody and minibody for clinical applications.

Genetically engineered radiolabeled antibody fragments have shown great promise for the radioimmunoscintigraphy of cancer. Retaining the exquisite specificity of monoclonal antibodies yet smaller in molecular size, antibody fragments display rapid tumor targeting and blood clearance, a more uniform distribution in the tumor, and present a lower potential to elicit an immune response. However, one of the factors that has limited clinical evaluation of these antibody-derived proteins has been the difficulty in expressing and purifying the quantities necessary for clinical trials. This study outlines the capability of mammalian expression for the production of recombinant antibody fragments intended for clinical use. Two anti-carcinoembryonic antigen antibody fragments, the T84.66/212 Flex minibody (scFv-C(H)3) and the T84.66 diabody (scFv dimer) have been previously expressed and have shown excellent radioimaging properties in tumor bearing animals. To proceed toward human studies, these high affinity recombinant fragments and a second minibody version, the T84.66/GS18 Flex minibody, were expressed using a high-level mammalian expression system. Production of all three antibody fragments in a small-scale hollow fiber bioreactor resulted in 137-307 mg of crude antibody harvest. A purification protocol that employed ceramic hydroxyapatite and anion exchange chromatography resulted in 50-150 mg of purified T84.66 diabody and T84.66 minibody. The development of this level of research grade material established conditions for clinical production as well as provided material to complete pre-clinical studies and undertake protein crystallization studies. Scale-up for clinical studies produced 3.4 g of the T84.66 minibody in the harvest. A portion of this material was purified yielding 180 mg of highly purified T84.66 minibody intended for pilot radioimmunoscintigraphy studies of carcinoembryonic antigen (CEA) positive disease.

MgATP binding to the nucleotide-binding domains of the eukaryotic cytoplasmic chaperonin induces conformational changes in the putative substrate-binding domains.

The eukaryotic cytosolic chaperonins are large heterooligomeric complexes with a cylindrical shape, resembling that of the homooligomeric bacterial counterpart, GroEL. In analogy to GroEL, changes in shape of the cytosolic chaperonin have been detected in the presence of MgATP using electron microscopy but, in contrast to the nucleotide-induced conformational changes in GroEL, no details are available about the specific nature of these changes. The present study identifies the structural regions of the cytosolic chaperonin that undergo conformational changes when MgATP binds to the nucleotide binding domains. It is shown that limited proteolysis with trypsin in the absence of MgATP cleaves each of the eight subunits approximately in half, generating two fragments of approximately 30 kDa. Using mass spectrometry (MS) and N-terminal sequence analysis, the cleavage is found to occur in a narrow span of the amino acid sequence, corresponding to the peptide binding regions of GroEL and to the helical protrusion, recently identified in the structure of the substrate binding domain of the archeal group II chaperonin. This proteolytic cleavage is prevented by MgATP but not by ATP in the absence of magnesium, ATP analogs (MgATPyS and MgAMP-PNP) or MgADP. These results suggest that, in analogy to GroEL, binding of MgATP to the nucleotide binding domains of the cytosolic chaperonin induces long range conformational changes in the polypeptide binding domains. It is postulated that despite their different subunit composition and substrate specificity, group I and group II chaperonins may share similar, functionally-important, conformational changes. Additional conformational changes are likely to involve a flexible helix-loop-helix motif, which is characteristic for all group II chaperonins.

Design and characterization of a multisite fluorescence energy-transfer system for protein folding studies: a steady-state and time-resolved study of yeast phosphoglycerate kinase.

A multisite distance-based fluorescence resonance energy-transfer assay system was developed for the study of protein folding reactions. Single- and double-cysteine substitution mutagenesis was utilized to place sulfhydryl residues throughout the tertiary structure of the bidomain enzyme yeast phosphoglycerate kinase (PGK). These reactive cysteines were covalently modified with extrinsic donor [5-[[2-(2-iodoacetamido)ethyl]amino]-1-naphthalenesulfonic acid] and acceptor (5-iodoacetamidofluorescein) fluorescent labels. A detailed experimental strategy was followed, which revealed that, when these relatively large extrinsic fluorescent labels are covalently attached to properly selected solvent-exposed residues, they do not affect the intrinsic stability of the protein. The PGK crystal structure was combined with molecular dynamics simulations of the dyes built into the protein and time-resolved anisotropy experiments, in order to estimate a more realistic orientation factor, *, for each donor/acceptor pair. Time-resolved and steady-state fluorescence energy-transfer experiments revealed that this distance assay, spanning six different donor-acceptor distances, is linear and accurate (to within 10-20%) over the range of 30-70 A. This distance assay system for PGK allows for the measurement of long-range changes in intra- and interdomain spatial organization during protein folding reactions. The approach which we have developed can be applied to any protein system in which unique one- and two-site cysteine residues can be engineered into a protein. In the following paper [Lillo, M. P., et al. (1997) Biochemistry 36, 11273-11281], these multisite energy-transfer pairs are utilized for stopped-flow unfolding studies.

Real-time measurement of multiple intramolecular distances during protein folding reactions: a multisite stopped-flow fluorescence energy-transfer study of yeast phosphoglycerate kinase.

Understanding the set of rules which dictate how the primary amino acid sequence determines tertiary structure is an unsolved problem in biophysics. If it were possible to simultaneously measure all of the intramolecular distances in a protein (in real time) during a folding reaction, the "second" genetic code problem would be solved. Regrettably, no such technique currently exists. As a first step toward this goal, an optical distance assay system has been developed for a two-domain protein, yeast phosphoglycerate kinase (PGK), using Förster resonance energy transfer [Lillo, M. P., et al. (1997) Biochemistry 36, 11261-11272]. In this study, real-time stopped-flow distance changes are measured using six unique pairs of donor/acceptor fluorescent labels strategically placed throughout the tertiary structure of PGK. These multiple donor/acceptor sites were genetically engineered into PGK by cysteine substitution mutagenesis followed by extrinsic labeling with fluorescent probes, 5-[[[(2-iodoacetyl)amino]ethyl]amino]naphthalenesulfonic acid (as a donor) and 5-iodoacetamidofluorescein (acceptor). The unfolding of PGK is found to be a sequential multistep process (native --> I1 --> I2 --> unfolded) with rate constants of 0.30, 0.16, and 0.052 s-1, respectively (from native to unfolded). Unique to this unfolding study, six intramolecular distance vectors have been resolved for both the I1 and I2 states. With this distance information, it is shown that the transition from the native to I1 state can be modeled as a large hinge-bending motion, in which both domains "swing away" from each other by about 15 A. As the domains move apart, the carboxyl-terminal domain rotates almost 90 degrees about the hinge region connecting the two domains. It is also shown that the amino-terminal domain remains intact during the native --> I1 transition, consistent with our previous site-specific tryptophan fluorescence anisotropy stopped-flow study [Beechem, J. M., et al. (1995) Biochemistry 34, 13943-13948]. Future experiments are proposed which will attempt to resolve in detail the unfolding/refolding transitions in this protein with a resolution of approximately 5-10 A.

Urea-induced equilibrium unfolding of single tryptophan mutants of yeast phosphoglycerate kinase: evidence for a stable intermediate.

Several single-tryptophan mutants of yeast phosphoglycerate kinase (PGK) have been used in the present study to characterize the urea-induced unfolding of PGK. A possibility that residual structures might be present in the urea-unfolded state was also investigated. The urea-induced unfolding transitions were monitored using circular dichroism (CD) and fluorescence techniques. The presence of stable intermediate(s) during urea-induced unfolding is suggested by biphasic transitions detected for the mutants containing tryptophans in the N-terminal domain and by the noncoincidence of transitions detected by various methods for other mutants. The N-terminal tryptophan probes exhibit hyperfluorescent properties in the intermediate state and a wavelength of maximum emission that lies between that of the native and unfolded state. This unfolding intermediate exhibits a major decrease in the ellipticity at 220 nm, but only a minor decrease at 278 nm, relative to the native state. These results suggest a significant loss of secondary structure content and a relatively small change in the asymmetric environment of tyrosine residues. Increased 1-anilinonaphthalene-8-sulfonic acid binding in the denaturant concentration range corresponding to the N --> I transition indicates the presence of a partially folded structure with exposed hydrophobic surfaces. These results demonstrate that the partially folded intermediates detected during urea-induced denaturation are structurally similar to those detected previously during guanidine-induced denaturation. No significant differences were detected between the urea- and guanidine-unfolded proteins on the basis of their fluorescence and CD properties.

Equilibrium unfolding of yeast phosphoglycerate kinase and its mutants lacking one or both native tryptophans: a circular dichroism and steady-state and time-resolved fluorescence study.

Yeast 3-phosphoglycerate kinase contains two tryptophans, both situated in the carboxy-terminal domain, and seven tyrosines, five in the amino-terminal domain, one in the domain-domain interface, and one in the carboxy-terminal domain. Site-specific mutagenesis has been used to construct two single-tryptophan mutants and one no-tryptophan mutant by replacing one or both native tryptophans, W308 and W333, with phenylalanines. The mutations have been shown to have a relatively small effect on the overall structure and enzymatic properties of the mutants. Both tryptophans are quenched in the folded state. The steady-state emission spectra and tryptophan quantum yields are the same in the single-tryptophan mutants and in the wild-type protein. Large changes in the tryptophan emission maxima and steady-state emission intensities are observed upon unfolding. Far-UV circular dichroism and steady-state as well as time-resolved fluorescence spectroscopy have been used to monitor the equilibrium unfolding transitions of these mutants and wild-type PGK. For each protein, the transitions followed by CD and steady-state fluorescence are nearly coincident, suggesting that the structural changes monitored by local fluorescence probes and ellipticity changes, which are sensitive to the changes in the overall structure, report a single cooperative transition, consistent with a two-state unfolding mechanism. Both tryptophans have three lifetimes, which follow a similar pattern as a function of denaturant concentration. The amplitude terms associated with the two longer lifetimes increase with unfolding while the short lifetime amplitude decreases. It thus appears that these population amplitudes represent markers for the unfolded and folded states, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Domain motions in phosphoglycerate kinase: determination of interdomain distance distributions by site-specific labeling and time-resolved fluorescence energy transfer.

3-Phosphoglycerate kinase is composed of two globular domains separated by a wide cleft. The substrate binding sites are situated on the inner surfaces of the two domains. By analogy to other kinases, it has been postulated that the catalytic mechanism of phosphoglycerate kinase involves a hinge bending domain motion that brings the substrates together to allow phosphoryl transfer. To characterize this large-scale conformational change, as well as the dynamics of the unliganded enzyme in solution, we have applied site-directed mutagenesis and time-resolved nonradiative energy transfer techniques. Two genetically engineered cysteines (Cys-135 and Cys-290), one in each of the two domains, were covalently labeled with a donor and acceptor pair of fluorescent probes. Analysis of subnanosecond fluorescence decay curves yielded the equilibrium distribution of interdomain distances. In the absence of substrates, the distribution of distances between the two labeled sites was very broad, with a full width at half maximum estimated as 20 A or broader, indicative of a large number of conformational substates in solution. The mean distance, 31.5 +/- 1 A, was 8 A smaller than in the crystal structure. Upon addition of ATP alone or of ATP and 3-phosphoglycerate, the average distance increased to 38 +/- 1 A and the width of the distribution decreased. Addition of 3-phosphoglycerate alone induced a similar but smaller change. The rate of conformational state fluctuations (interconversion between states) was found to be slow on the nanosecond time scale, as expected for a protein with a relatively large interdomain contact area.

Probing the role of arginines and histidines in the catalytic function and activation of yeast 3-phosphoglycerate kinase by site-directed mutagenesis.

A cluster of conserved histidines and arginines (His-62, His-167, Arg-21, Arg-38, and Arg-168) in 3-phosphoglycerate kinase (PGK) has been implicated as possibly involved in the binding of 3-phosphoglycerate (3-PG) and/or stabilization of the negatively charged transition state. The role of these residues in the catalytic function of yeast PGK and in the substrate- and sulfate-dependent activation was investigated by site-directed mutagenesis. The following substitutions, R21A, R21Q, H62Q, H167S, and R168Q, produced functional enzymes. In contrast, the R38A and R38Q mutations resulted in a complete loss of catalytic activity. These results demonstrate that of the basic residues studied, only arginine 38 is essential for the catalytic function of PGK. A moderate decrease in the catalytic efficiency as the result of the R21A, H167S, and R168Q mutations and an increased catalytic efficiency of the H62Q mutant rule out a possible role of a positive charge at these positions in the mechanism of phosphoryl transfer reaction. In contrast to the wild type PGK and the H62Q mutant, both of which are activated at low and inhibited at high sulfate concentration, the H167S, R168Q, and R21A mutants exhibited a progressive inhibition with increased concentration of sulfate. The activation observed at high concentration of either ATP or 3-PG as a variable substrate in the steady-state kinetics of wild type PGK was abolished as the result of the latter three mutations. The results of this work support the hypothesis that PGK has two binding sites for anionic ligands, the catalytic and regulatory sites for each substrate and the activatory and inhibitory sites for sulfate, and suggest that arginine 21, arginine 168, and histidine 167 are located in the activatory anion binding site, common for sulfate, 3-PG, and ATP. The increased Km values for both substrates and decreased specific activities of the mutants suggest that this regulatory site is close to the catalytic site.