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The conjugation of polyethylene glycol (PEGylation) with downsized compact antibodies is an effective method for overcoming the problem of rapid elimination of the compact antibodies from the body. We integrated site-specific PEGylation with the refolding of a single-chain Fv (scFv) of humanized monoclonal antibody 528 with affinity for the epidermal growth factor receptor, to prepare active PEGylated scFv from insoluble aggregates produced in an Escherichia coli expression system. The insertion of a cysteine residue at the C-terminus of scFv to serve as the conjugation site for PEG led to the formation of highly multimeric scFv during the refolding process; however, PEGylation after refolding drastically dispersed the multimer into monomeric active scFv fragments. Further, the PEGylation of partially refolded scFv during the refolding process improved the PEGylation efficiency and suppressed the formation of highly multimeric scFv; consequently, monomeric active scFv fragments were obtained directly from the insoluble aggregates in E. coli. We show that in vitro refolding of PEGylated scFv should be useful for improving downstream processing performance in the production of clinically useful small antibodies from insoluble fractions., ((c) 2009 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

We report the preparation and functional characterization of an Escherichia coli-expressed recombinant fusion protein, 528scFv-TRAIL, specific for the human epidermal growth factor receptor (EGFR) and empowered by the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). The 528scFv-TRAIL, expressed as insoluble inclusion bodies in E. coli, was solubilized and then refolded by using a modified stepwise dialysis method. Treatment with 528scFv-TRAIL resulted in the specific binding to the cell surface of EGFR-positive cells with concomitant deployment of the TRAIL moiety to DR-5 receptor in a manner comparable to a commercially available form of recombinant TRAIL (cTRAIL). 528scFv-TRAIL, prepared by either of three refolding processes described herein, showed potent cytotoxic activity against EGFR-positive TFK-1 cell line and was superior to its parental 528scFv; a recombinant variable fragment with single specificity against human EGFR. Narrow variations in the cytotoxic potential of 528scFv-TRAIL were ascribed to manipulation of redox conditions during the refolding process. Together, our findings point to the potential value of 528scFv-TRAIL for treatment of EGFR-expressing cancers. Furthermore, preparation of 528scFv-TRAIL from insoluble aggregates in a prokaryotic cell based expression system by means of in vitro refolding introduces a feasible cost-benefit, time-efficient approach for industrial-scale production.

A microbial array chip with collagen gel spots entrapping living Escherichia coli (E. coli) DH5alpha was applied for the screening of recombinant protein solubilities. The alpha-fragment of beta-galactosidase (betaGal) was fused to the target protein, namely, maltose-binding protein (MBP), to monitor the solubility of MBP. Scanning electrochemical microscopy (SECM) was used to detect the release of p-aminophenol from E. coli cells catalyzed by intracellular betaGal. Comparison of the SECM-based method with the Western blotting-based method indicated that the current response obtained using SECM increased with an increase in the betaGal activity and therefore, with the soluble fraction of MBP in the host cells., ((c) 2006 Wiley Periodicals, Inc.)

Expression of beta2 microglobulin (beta2m) in Escherichia coli resulted in formation of inclusion bodies. Attenuated total reflectance Fourier transform infrared analysis suggested a native-like secondary structure of beta2m in the inclusion bodies. Nondenaturing solubilization of the native-like beta2m from inclusion bodies was achieved using L-arginine solution, which enables an efficient recovery of beta2m with little aggregation. Greater beta2m solubilization from inclusion bodies was obtained at higher temperatures. Low-temperature solubilization yielded beta2m with fluorescence properties identical to those of native beta2m, but its secondary structure was slightly nonnative. Solubilization at moderate temperature gave beta2m with an apparently native structure. We propose an efficient nondenaturing solubilization method combining L-arginine and moderate temperature.

Expression of green fluorescent protein (GFP) in Escherichia coli (E. coli) resulted in only small amount of soluble and fluorescent GFP protein and hence most of the protein in insoluble particles. The expressed GFP in insoluble particles, however, was fluorescent, indicating that it is at least in part folded with an intact chromophore. The GFP in insoluble particles could not be solubilized by an aqueous (denaturant-free) buffer. Solubilization of active GFP from insoluble particles was then attempted with guanidine hydrochloride (GdnHCl), a strong protein-denaturant, or L-arginine, an aggregation suppressor. Solubilization from insoluble particles by 6M GdnHCl led to complete denaturation of the GFP existing in insoluble particles, while GdnHCl solution at lower concentration could solubilize fluorescent GFP. Solubilization of fluorescently active GFP from insoluble particles was also achieved by L-arginine. It is noteworthy that L-arginine was stronger in solubilizing insoluble GFP than GdnHCl below 2M. These results demonstrate that some proteins expressed in E. coli may form insoluble particles containing native conformation and L-arginine may be used to recover the proteins in the native form from such insoluble particles.

Interleukin-15 receptor alpha-chain (IL-15Ralpha) is a member of the new cytokine receptor family, which possesses the sushi domain. To investigate the biochemical and biophysical characteristics of soluble human IL-15Ralpha (shIL-15Ralpha), shIL-15Ralpha was recombinantly expressed in Escherichia coli. The shIL-15Ralpha containing a six histidine-tag was expressed as inclusion bodies, which were solubilized with urea, immobilized on a Ni-nitrilotriacetic acid column, and refolded by a decreasing gradient of urea concentration. The refolded shIL-15Ralpha exhibited a highly flexible structure, neutralized human interleukin-15-induced cell proliferation effectively, and bound to its ligand with the same affinity as human IL-15Ralpha on the cell surface, as demonstrated by circular dichroism, a cell proliferation assay, and surface plasmon resonance, respectively. Thus, we succeeded in refolding shIL-15Ralpha to an active form on an affinity column.

Using biotinylated phage (BIO-phages), we observed the infection of filamentous phages into Escherichia coli JM109 morphologically. BIO-phages and BIO-phage-derived proteins, mainly pVIII, were detected in E. coli by using the avidin-biotin-peroxidase complex method with electron microscopy. Infected cells revealed positive staining on the outer and inner membranes and in the periplasmic space. Some cells showed specific or predominant staining of the outer membrane, whereas others showed predominant staining of the inner membrane or equivalent staining of the outer and inner membranes. The periplasmic spaces in some infected cells were expanded and filled with reaction products. Some cells showed wavy lines of positive staining in the periplasmic space. BIO-phages were detected as thick filaments or clusters covered with reaction products. The ends of the infecting phages were located on the surface of cells, in the periplasmic space, or on the inner membrane. These findings suggest that phage major coat proteins are integrated into the outer membrane and that phages cause periplasmic expansion during infection.

Several recombinant proteins in inclusion bodies expressed in Escherichia coli have been measured by Fourier transform infrared and solid-state nuclear magnetic resonance spectra to provide the secondary structural characteristics of the proteins from hyperthermophilic archaeon Pyrococcus horikoshii OT3 (hyperthermophilic proteins) in inclusion bodies. The β-strand-rich single chain Fv fragment (scFv) and α-helix-rich interleukin (IL)-4 lost part of the native-like secondary structure in inclusion bodies, while the inclusion bodies composed of the hyperthermophilic proteins of which the native form is α-helix rich, are predominated by α-helix structure. Further, the secondary structure of the recombinant proteins solubilized from inclusion bodies by detergent or denaturant was observed by circular dichroism (CD) spectra. The solubilization induced the denaturation of the secondary structure for scFv and IL-4, whereas the solubilized hyperthermophilic proteins have retained the α-helix structure with the CD properties resembling those of their native forms. This indicates that the hyperthermophilic proteins form native-like secondary structure in inclusion bodies. Refolding of several hyperthermophilic proteins from in vivo aggregated form without complete denaturation could be accomplished by solubilization with lower concentration (e.g. 2 M) of guanidine hydrochloride and removal of the denaturant via stepwise dialysis. This supports the existence of proteins with native-like structure in inclusion bodies and suggests that non-native association between the secondary structure elements leads to in vivo aggregation. We propose a refolding procedure on the basis of the structural properties of the aggregated archaeon proteins. [Copyright &y& Elsevier]

We have been able to isolate several species of 5-S ribosomal RNA from Eschzerichia coli A19. These molecules were separated on the basis of their differing stabilities during electrophoresis on 12% polyacrylamide gels in 7 M urea. This differing stability is shown, in one case, to be due to a different primary sequence. We have determined the sequence of the least stable of these molecules and have found only one difference to the published sequence of E. coli A19 5-S RNA, namely a uridine in place of a cytidine at position 92. The consequent G U base pair, formed in a normally highly stable G. C-rich region. is responsible for a drastic reduction in the stability of the molecule. This instability leads to a less constrained, more compact molecule which thus migrates laster in electrophoresis under denaturing conditions. This species of 5-S RNA is shown to make up 30% of the total 5-S RNA in the 50-S ribosomal subunits in this organism. Further structural studies were carried out using S1 nuclease digestion, sodium bisulphite modification and thermal melting analysis. All these methods indicate a 5-S RNA drastically destabilized in parts of its secondary and tertiary structure. Finally, the ability of the variant 5-S RNA to recognize and form a complex with its 50-S subunit binding proteins was examined and found to be impaired. [ABSTRACT FROM AUTHOR]

The gene encoding a flavodoxin of Desulfovibrio vulgaris (Miyazaki F) was cloned, and overexpressed in Escherichia coli. A 1.6-kbp DNA fragment, isolated from D. vulgaris (Miyazaki F) by double digestion with SalI and EcoRI, contained the flavodoxin gene and its regulatory region. An expression system for the flavodoxin gene under control of the T7 promoter was constructed in E. coli. The purified protein was soluble and exhibited a characteristic visible absorption spectrum. HPLC analysis of the recombinant flavodoxin revealed the presence of an identical FMN to that found in the native D. vulgaris flavodoxin, and its dissociation constant with FMN was determined to be 0.38 nM. In vitro H2 reduction analysis indicated that the recombinant flavodoxin is active, and its redox potential was determined to be E1 = −434 and E2 = −151 mV using methyl viologen and 2-hydroxy-1,4-naphthoquinone, respectively. Its redox behavior was also examined with the recombinant flavodoxin adsorbed onto a graphite electrode. The mutant, A16E, was also produced, which revealed the feature of a conserved Glu residue at the surface of the molecule. [ABSTRACT FROM AUTHOR]

The cytotoxicity of T cell-recruiting antibodies with their potential to damage late-stage tumor masses is critically dependent on their structural and functional properties. Recently, we reported a semi-high-throughput process for screening highly cytotoxic small bispecific antibodies (i.e., diabodies). In the present study, we improved the high-throughput performance of this screening process by removing the protein purification stage and adding a stage for determining the concentrations of the diabodies in culture supernatant. The diabodies were constructed by using an Escherichia coli expression system, and each diabody contained tandemly arranged peptide tags at the C-terminus, which allowed the concentration of diabodies in the culture supernatant to be quantified by using a tag-sandwich enzyme-linked immunosorbent assay. When estimated diabody concentrations were used to determine the cytotoxicity of unpurified antibodies, results comparable to those of purified antibodies were obtained. In a surface plasmon resonance spectroscopy-based target-binding assay, contaminants in the culture supernatant prevented us from conducting a quantitative binding analysis; however, this approach did allow relative binding affinity to be determined, and the relative binding affinities of the unpurified diabodies were comparable to those of the purified antibodies. Thus, we present here an improved high-throughput process for the simultaneous screening and determination of the binding parameters of highly cytotoxic bispecific antibodies. [ABSTRACT FROM AUTHOR]

Abstract: The electromotility of outer hair cells (OHCs) is believed to be a major factor in cochlear amplification that enables the high sensitivity of hearing in mammals. This motility is thought to be based on voltage-dependent conformational changes of a motor protein embedded in the lateral wall of the OHC. In 2000, this motor protein was identified and termed prestin. To obtain knowledge on the function of prestin, research at the molecular level is necessary. For this purpose, a method of obtaining a large amount of prestin is required. In this study, an attempt was therefore made to construct an expression system for prestin. Prestin cDNA was introduced into Escherichia coli (E. coli), insect cells and Chinese hamster ovary (CHO) cells, and the expression of prestin was examined by Western blotting. As CHO cells expressed prestin well, we generated prestin-expressing cell lines using CHO cells by limiting dilution cloning. The stable expression and the activity of prestin in generated cell lines were then confirmed. Finally, to obtain prestin from these cell lines efficiently, culture conditions of the cells were examined, and it was clarified that cells should be cultured in serum-free medium and harvested around 48h after passage. [Copyright &y& Elsevier]

Abstract: Integration of lymphocyte-activating cytokines (e.g., interleukin-12: IL-12) to tumor cells offers promise for cancer immunotherapy, but the preparation of such heterodimeric proteins by refolding is difficult because of subunit instability. We achieved the refolding of Escherichia coli-expressed human IL-12 by a stepwise dialysis method, preventing the formation of insoluble aggregates by adding a redox reagent and an aggregation suppressor. We also constructed a tumor-specific IL-12 protein, each subunit of which was fused with one chain of variable domain fragment (Fv) of anticarcinoembryonic antigen (CEA) antibody T84.66 (aCEA-IL12). Fusion of IL-12 with Fv greatly increased the yield of functional heterodimer. Several assays have indicated that the Fv domain and IL-12 domain of the fused protein had cognate biological activities, and it enhanced the cytotoxicity of T-LAK cells for the cancer cell line. [Copyright &y& Elsevier]

• Nearly half the LH diabody fraction and all the HL fraction were in inactive form. • Diabody fraction components changed dynamically with time. • The association of diabodies corresponded to that of the scFv chimera. • Addition of the deficient HL-R chimera scFV increased cancer cell cytotoxicity. Diabody 31, a bispecific therapeutic antibody, is a heterodimer comprised of two types of chimeric single-chain variable fragments (scFv), that has been identified as a clone with high cytotoxicity against cancer cells. Diabody 31 is inactive as a homodimer and monomer, yet active as a heterodimer in solution. Herein, we examined the association between two kinds of diabodies, LH- and HL type, based on the behavior of four kinds of their constituent chimera scFvs, by size analysis. More than half of the LH diabody fraction, including two types of chimeric scFV that were equally expressed, and nearly all HL diabody fraction components, including one chimeric HL-L scFv, were in inactive form and changed components dynamically as time passed. The association of these diabodies corresponded to that of the scFv chimera, indicating that analysis of these chimeras could predict diabody quality. Furthermore, addition of the deficient HL-R chimera scFv, comprised of an unbalanced chimeric scFv ratio, to the HL diabody fraction increased cancer cell cytotoxicity, with maximal effects obtained upon repeating the process five times. These findings provide insights into the efficient construction of a functional diabody by noting the characteristics of associated diabodies and the molar ratio of expressed chimera scFvs. [ABSTRACT FROM AUTHOR]