Lenticular chaperones suppress the aggregation of the cataract-causing mutant T5P gamma C-crystallin

The T5P mutation in human gamma C-crystallin produces a lens cataract. Here, we have investigated the effects of the T5P mutation upon the aggregation of gamma C-crystallin in vitro and in transfected cells. By sedimentation assay and sucrose gradient centrifugation, the mutation significantly increased the aggregation of the protein and reduced dramatically its solubility in vitro. Similar effects were seen when T5P gamma C-crystallin was transfected into tissue culture cells, resulting in the formation of cytoplasmic aggregates of T5P gamma C-crystallin. Interestingly, the major lenticular protein chaperones, alpha A- and alpha B-crystallin, increased the solubility of the T5P gamma C-crystallin both in vitro and in transfected cells. More importantly, the size of the T5P gamma C-crystallin aggregates were also significantly reduced in the presence of the lenticular chaperones. These data therefore suggest a dual role for these chaperones in maintaining transparency in the lens. The first is that these protein chaperones increase the proportion of the soluble T5P gamma C-crystallin and the second is that they also reduce light scatter by reducing the aggregate size of T5P gamma C-crystallin. Both activities could modify the cataract phenotype and help explain the observed variability reported for identical gamma-crystallin mutations, which identify cataract as a polygenic disease.     

The osmolyte betaine promotes protein misfolding and disruption of protein aggregates

In this work the effect of betaine on the structure and aggregation of the GST-GFP fluorescent fusion protein was studied by different complementary techniques, including electron microscopy, dynamic light scattering, circular dichroism, and FTIR spectroscopy. Although osmolytes are known to be protein stabilizers in vivo, the effect of betaine on the structure and aggregation of our model protein was found to be strictly concentration dependent. We demonstrated that, by changing betaine concentration, it was possible to tune the formation of protein soluble assemblies and insoluble aggregates, as well as to disaggregate preformed aggregates. In particular, at a critical concentration of betaine between 5 and 7.5 mM, the protein precipitated into macroscopic prefibrillar structures, rich in intermolecular beta-sheets, which were found to bind thioflavine T and to be inaccessible to protease. Instead, at higher betaine concentration (10-20 mM) the misfolded protein lost its fluorescence, but formed soluble assemblies with hydrodynamic radius of about 16 nm. These structures displayed a reduced propensity to further aggregate under thermal treatment. In addition, betaine at this high concentration was also found to disrupt large preformed aggregates, obtained under different conditions, into protein soluble assemblies. It is the first time that a disaggregation process has been described for a chemical chaperone. A mechanism for the betaine concentration-dependent effect on protein misfolding, aggregation, and disaggregation is proposed and its possible physiological implications are discussed.     

A favorable solubility partner for the recombinant expression of streptavidin

Recombinant streptavidin is extremely difficult to express at high levels in the cytoplasm of Escherichia coli without the formation of inclusion bodies. Fusing a solubility enhancing partner to an aggregation prone protein is a widely used tool to circumvent inclusion body formation. Here, we use streptavidin as a target protein to test the properties of N-terminal fragments of translation initiation factor IF2 from E. coli as a solubility partner. Domain I (residue 1-158) of IF2 is superior to the well-established solubility partners maltose-binding protein (MBP) and NusA for soluble expression of active streptavidin. The number of active streptavidin molecules isolated by chromatography is increased threefold when domain I is used as solubility partner as compared to MBP or NusA. The relatively small size, high expressivity, and extreme solubility make domain I of IF2 an ideal partner for streptavidin and may also prevent other recombinant proteins such as ScFv antibodies from being expressed as insoluble aggregates in the cytoplasm of E. coli.     

Properties and growth mechanism of the ordered aggregation of a model RNA by the HIV-1 nucleocapsid protein: An electron microscopy investigation

NCp7, the nucleocapsid protein of the human immunodeficiency virus type 1, induces an ordered aggregation of RNAs, a mechanism that is thought to be involved in the NCp7-induced promotion of nucleic acid annealing. To further investigate this aggregation, the morphology and the properties of the NCp7-induced aggregates of the model RNA homoribopolymer, polyA, were investigated by electron microscopy in various conditions. In almost all the tested conditions, the aggregates were spherical and consisted of a central dense core surrounded by a less dense halo made of NCp7-covered polyA molecules. The formation of these aggregates with a narrow distribution of sizes constitutes a distinctive feature of NCp7 over other single-stranded nucleic acid binding proteins. In most conditions, at the shortest times that can be reached experimentally, all the polyA molecules were already incorporated in small aggregates, suggesting that the nucleation step and the first aggregation events took place rapidly. The aggregates then orderly grew with time by fusion of the smaller aggregates to give larger ones. The aggregate halo was important in the fusion process by initiating the bridging between the colliding aggregates. In the presence of an excess of protein, the aggregates grew rapidly but were loosely packed and dissociated easily, suggesting adverse protein-protein interactions in the aggregates obtained in these conditions. In the presence of an excess of nucleotides, the presence of both amorphous nonspherical and slowly growing spherical aggregates suggested some changes in the mechanism of aggregate growth due to an incomplete covering of polyA molecules by NCp7. Finally, we showed that in the absence of added salt, the aggregate fusions were unfavored but not the initial events giving the first aggregates, the reverse being true in the presence of high salt concentrations (≥300 mM). © 1998 John Wiley & Sons, Inc. Biopoly 45: 217–229, 1998     

Impact of Cavitation,High Shear Stress and Air/Liquid Interfaces on Protein Aggregation

The reported impact of shear stress on protein aggregation has been contradictory. At high shear rates, the occurrence of cavitation or entrapment of air is reasonable and their effects possibly misattributed to shear stress. Nine different proteins (α‐lactalbumin, two antibodies, fibroblast growth factor 2, granulocyte colony stimulating factor [GCSF], green fluorescence protein [GFP], hemoglobin, human serum albumin, and lysozyme) are tested for their aggregation behavior on vapor/liquid interfaces generated by cavitation and compared it to the isolated effects of high shear stress and air/liquid interfaces generated by foaming. Cavitation induced the aggregation of GCSF by +68.9%, hemoglobin +4%, and human serum albumin +2.9%, compared to a control, whereas the other proteins do not aggregate. The protein aggregation behaviors of the different proteins at air/liquid interfaces are similar to cavitation, but the effect is more pronounced. Air‐liquid interface induced the aggregation of GCSF by +94.5%, hemoglobin +35.5%, and human serum albumin (HSA) +31.1%. The results indicate that the sensitivity of a certain protein toward cavitation is very similar to air/liquid‐induced aggregation. Hence, hydroxyl radicals cannot be seen as the driving force for protein aggregation when cavitation occurs. Further, high shear rates of up to 10⁸ s^?1 do not affect any of the tested proteins. Therefore, also within this study generated extremely high isolated shear rates cannot be considered to harm structural integrity when processing proteins.

     

Dimorphic aggregation behavior of a fusion polypeptide incorporating a stable protein domain (EGFP) with an amyloidogenic sequence (retroCspA)

We describe the behavior of a polypeptide consisting of the genetic fusion of a structurally stable single-domain protein, EGFP (an analog of the green fluorescent protein) with an amyloidogenic sequence, retroCspA (known to readily form amyloid fibrils). Refolding of the fusion protein through single-step removal of denaturant and salt results in precipitation into amyloid aggregates displaying fibrillar morphology, thioflavin T binding as well as green fluorescence. Refolding through step-wise reduction of denaturant concentration in the presence of salt yields a soluble aggregate containing a folded, thermally-stable, non-fluorescent EGFP domain. Together, these results indicate that retroCspA forces the fusion protein to aggregate; however, the EGFP domain remains folded in a native-like structural format in both soluble aggregates and precipitates.     

Conformation and self-association of human recombinant transforming growth factor-beta3 in aqueous solutions

The transforming growth factors-beta (TGF-beta) are important regulatory peptides for cell growth and differentiation with therapeutic potential for wound healing. Among the several TGF-beta isoforms TGF-beta3 has a particularly low solubility at physiological pH and easily forms aggregates. A spectroscopic structural analysis of TGF-beta3 in solution has thus been difficult. In this study, circular dichroism spectroscopy was used to determine the secondary structural elements of TGF-beta3. In addition, the aggregation of TGF-beta3 was investigated systematically as a function of pH and salt concentration using a rapid screening method. Sedimentation equilibrium and sedimentation velocity analysis revealed that TGF-beta3 exists predominantly in two major forms: (i) monomers in solution at low pH and (ii) large precipitating aggregates at physiological pH. Under acidic conditions (pH < 3.8) the protein was not aggregated. At pH approximately 3.9, a monomer right arrow over left arrow dimer equilibrium could be detected that transformed into larger aggregates at pH > 4.1. Aggregation was pronounced in the pH range of 4.3 < pH < 9.8 with the aggregation maximum between pH 6.5 and 8. 5. The aggregation process was accompanied by a structural change of the protein. The CD spectra were characterized by an isodichroic point at 209.5 nm indicating a two-state equilibrium between TGF-beta3 dissolved in solution and aggregated TGF-beta3. Aggregated TGF-beta3 showed a higher beta-sheet content and lower beta-turn and random coil contributions compared with monomeric TGF-beta3. Both the solution structure and the aggregate structure of TGF-beta3 were different from the crystal structure. This was in contrast to TGF-beta2, which showed very similar crystal and solution structures. Under alkaline conditions (pH > 9.8) the turbidity disappeared and a further conformational change was induced. The pH dependence of the TGF-beta3 conformation in solution in the range of 2.3 < pH < 11. 0 was reversible. Aggregation of TGF-beta3 was, furthermore, influenced by the presence of salt. For pH > 3.8 the addition of salt greatly enhanced the tendency to aggregate, even in the very basic domain. Under physiological conditions (pH 7.4, cNaCl = 164 mM) TGF-beta3 has almost the highest tendency to aggregate and will remain in solution only at nanomolar concentrations.     

Mechanism of the antichaperone activity of protein disulfide isomerase: facilitated assembly of large, insoluble aggregates of denatured lysozyme and PDI

Protein disulfide isomerase (PDI), a folding catalyst and chaperone can, under certain conditions, facilitate the misfolding and aggregation of its substrates. This behavior, termed antichaperone activity [Puig, A., and Gilbert, H. F., (1994) J. Biol. Chem. 269, 25889] may provide a common mechanism for aggregate formation in the cell, both as a normal consequence of cell function or as a consequence of disease. When diluted from the denaturant, reduced, denatured lysozyme (10-50 microM) remains soluble, although it does aggregate to form an ensemble of species with an average sedimentation coefficient of 23 +/- 5 S (approximately 600 +/- 100 kDa). When low concentrations of PDI (1-5 microM) are present, the majority (80 +/- 8%) of lysozyme molecules precipitate in large, insoluble aggregates, together with 87 +/- 12% of the PDI. PDI-facilitated aggregation occurs even when disulfide formation is precluded by the presence of dithiothreitol (10 mM). Maximal lysozyme-PDI precipitation occurs at a constant lysozyme/PDI ratio of 10:1 over a range of lysozyme concentrations (10-50 microM). Concomitant resolubilization of PDI and lysozyme from these aggregates by increasing concentrations of urea suggests that PDI is an integral component of the mixed aggregate. PDI induces lysozyme aggregation by noncovalently cross-linking 23 S lysozyme species to form aggregates that become so large (approximately 38,000 S) that they are cleared from the analytical ultracentrifuge even at low speed (1500 rpm). The rate of insoluble aggregate formation increases with increasing PDI concentration (although a threshold PDI concentration is observed). However, increasing lysozyme concentration slows the rate of aggregation, presumably by depleting PDI from solution. A simple mechanism is proposed that accounts for these unusual aggregation kinetics as well as the switch between antichaperone and chaperone behavior observed at higher concentrations of PDI.     

Aggregation kinetics of interrupted polyglutamine peptides

Abnormally expanded polyglutamine domains are associated with at least nine neurodegenerative diseases, including Huntington's disease. Expansion of the glutamine region facilitates aggregation of the impacted protein, and aggregation has been linked to neurotoxicity. Studies of synthetic peptides have contributed substantially to our understanding of the mechanism of aggregation because the underlying biophysics of polyglutamine-mediated association can be probed independent of their context within a larger protein. In this report, interrupting residues were inserted into polyglutamine peptides (Q20), and the impact on conformational and aggregation properties was examined. A peptide with two alanine residues formed laterally aligned fibrillar aggregates that were similar to the uninterrupted Q20 peptide. Insertion of two proline residues resulted in soluble, nonfibrillar aggregates, which did not mature into insoluble aggregates. In contrast, insertion of a β-turn template _DPG rapidly accelerated aggregation and resulted in a fibrillar aggregate morphology with little lateral alignment between fibrils. These results are interpreted to indicate that (a) long-range nonspecific interactions lead to the formation of soluble oligomers, while maturation of oligomers into fibrils requires conformational conversion and (b) that soluble oligomers dynamically interact with each other, while insoluble aggregates are relatively inert. Kinetic analysis revealed that the increase in aggregation caused by the _DPG insert is inconsistent with the nucleation-elongation mechanism of aggregation featuring a monomeric β-sheet nucleus. Rather, the data support a mechanism of polyglutamine aggregation by which monomers associate into soluble oligomers, which then undergo slow structural rearrangement to form sedimentable aggregates.     

Denatured Mammalian Protein Mixtures Exhibit Unusually High Solubility in Nucleic Acid-Free Pure Water

Preventing protein aggregation is a major goal of biotechnology. Since protein aggregates are mainly comprised of unfolded proteins, protecting against denaturation is likely to assist solubility in an aqueous medium. Contrary to this concept, we found denatured total cellular protein mixture from mammalian cell kept high solubility in pure water when the mixture was nucleic acids free. The lysates were prepared from total cellular protein pellet extracted by using guanidinium thiocyanate-phenol-chloroform mixture of TRIzol, denatured and reduced total protein mixtures remained soluble after extensive dialysis against pure water. The total cell protein lysates contained fully disordered proteins that readily formed large aggregates upon contact with nucleic acids or salts. These findings suggested that the highly flexible mixtures of disordered proteins, which have fully ionized side chains, are protected against aggregation. Interestingly, this unusual solubility is characteristic of protein mixtures from higher eukaryotes, whereas most prokaryotic protein mixtures were aggregated under identical conditions. This unusual solubility of unfolded protein mixtures could have implications for the study of intrinsically disordered proteins in a variety of cells.     

Reversibility of scrapie-associated prion protein aggregation

During the course of the transmissible spongiform encephalopathy diseases, a protease-resistant ordered aggregate of scrapie prion protein (PrP(Sc)) accumulates in affected animals. From mechanistic and therapeutic points of view, it is relevant to determine the extent to which PrP(Sc) formation and aggregation are reversible. PrP(Sc) solubilized with 5 m guanidine hydrochloride (GdnHCl) was unfolded to a predominantly random coil conformation. Upon dilution of GdnHCl, PrP refolded into a conformation that was high in alpha-helix as measured by CD spectroscopy, similar to the normal cellular isoform of PrP (PrP(C)). This provided evidence that PrP(Sc) can be induced to revert to a PrP(C)-like conformation with a strong denaturant. To examine the reversibility of PrP(Sc) formation and aggregation under more physiological conditions, PrP(Sc) aggregates were washed and resuspended in buffers lacking GdnHCl and monitored over time for the appearance of soluble PrP. No dissociation of PrP from the PrP(Sc) aggregates was detected in aqueous buffers at pH 6 and 7.5. The effective solubility of PrP was <0.7 nm. Treatment of PrP(Sc) with proteinase K (PK) before the analysis did not enhance the dissociation of PrP from the PrP(Sc) aggregates. Treatment with 2.5 m GdnHCl, which partially and reversibly unfolds PrP(Sc), caused only limited dissociation of PrP from the aggregates. The PrP that dissociated from the aggregates over time was entirely PK-sensitive, like PrP(C), whereas all of the aggregated PrP was partially PK-resistant. PrP also dissociated from aggregates of protease-resistant PrP generated in a cell-free conversion reaction, but only if treated with GdnHCl. Overall, the results suggest that PrP aggregation is not appreciably reversible under physiological conditions, but dissociation and refolding can be enhanced by treatments with GdnHCl.     

Proteins that mediate protein aggregation and cytotoxicity distinguish Alzheimer's hippocampus from normal controls

Neurodegenerative diseases are distinguished by characteristic protein aggregates initiated by disease‐specific ‘seed’ proteins; however, roles of other co‐aggregated proteins remain largely unexplored. Compact hippocampal aggregates were purified from Alzheimer's and control‐subject pools using magnetic‐bead immunoaffinity pulldowns. Their components were fractionated by electrophoretic mobility and analyzed by high‐resolution proteomics. Although total detergent‐insoluble aggregates from Alzheimer's and controls had similar protein content, within the fractions isolated by tau or Aβ_1–42 pulldown, the protein constituents of Alzheimer‐derived aggregates were more abundant, diverse, and post‐translationally modified than those from controls. Tau‐ and Aβ‐containing aggregates were distinguished by multiple components, and yet shared >90% of their protein constituents, implying similar accretion mechanisms. Alzheimer‐specific protein enrichment in tau‐containing aggregates was corroborated for individuals by three analyses. Five proteins inferred to co‐aggregate with tau were confirmed by precise in situ methods, including proximity ligation amplification that requires co‐localization within 40 nm. Nematode orthologs of 21 proteins, which showed Alzheimer‐specific enrichment in tau‐containing aggregates, were assessed for aggregation‐promoting roles in C. elegans by RNA‐interference ‘knockdown’. Fifteen knockdowns (71%) rescued paralysis of worms expressing muscle Aβ, and 12 (57%) rescued chemotaxis disrupted by neuronal Aβ expression. Proteins identified in compact human aggregates, bound by antibody to total tau, were thus shown to play causal roles in aggregation based on nematode models triggered by Aβ_1–42. These observations imply shared mechanisms driving both types of aggregation, and/or aggregate‐mediated cross‐talk between tau and Aβ. Knowledge of protein components that promote protein accrual in diverse aggregate types implicates common mechanisms and identifies novel targets for drug intervention.     

Aggregation of a multidomain protein: A coagulation mechanism governs aggregation of a model IgG1 antibody under weak thermal stress

Using an IgG1 antibody as a model system, we have studied the mechanisms by which multidomain proteins aggregate at physiological pH when incubated at temperatures just below their lowest thermal transition. In this temperature interval, only minor changes to the protein conformation are observed. Light scattering consistently showed two coupled phases: an initial fast phase followed by several hours of exponential growth of the scattered intensity. This is the exact opposite of the lag‐time behavior typically observed in protein fibrillation. Dynamic light scattering showed the rapid formation of an aggregate species with a hydrodynamic radius of about 25 nm, which then increased in size throughout the experiment. Theoretical analysis of our light scattering data showed that the aggregate number density goes through a maximum in time providing compelling evidence for a coagulation mechanism in which aggregates fuse together. Both the analysis as well as size‐exclusion chromatography of incubated samples showed the actual increase in aggregate mass to be linear and reach saturation long before all molecules had been converted to aggregates. The CH2 domain is the only domain partly unfolded in the temperature interval studied, suggesting a pivotal role of this least stable domain in the aggregation process. Our results show that for multidomain proteins at temperatures below their thermal denaturation, transient unfolding of a single domain can prime the molecule for aggregation, and that the formation of large aggregates is driven by coagulation.     

In vivo aggregation of a single enzyme limits growth of Escherichia coli at elevated temperatures

The formation of protein aggregates is associated with unfolding and denaturation of proteins. Recent studies have indicated that, in Escherichia coli, cellular proteins tend to aggregate when the bacteria are exposed to thermal stress. Here, we show that the aggregation of one single E. coli cytoplasmic protein limits growth at elevated temperatures in minimal media. Homoserine trans-succinylase (HTS), the first enzyme in the methionine biosynthetic pathway, aggregates at temperatures higher than 44 degrees C in vitro. Above this temperature, we can also observe in vivo aggregation that results in the complete disappearance of the enzyme from the soluble fraction. Moreover, reducing the in vivo level of HTS aggregation enables growth at non-permissive temperatures. This is the first demonstration of the physiological role of aggregation of a specific protein in the growth of wild-type bacteria.     

Characterization of full-length p53 aggregates and their kinetics of formation

Mutations in the TP53 gene are common in cancer with the R248Q missense mutation conferring an increased propensity to aggregate. Previous p53 aggregation studies showed that, at micromolar concentrations, protein unfolding to produce aggregation-prone species is the rate-determining step. Here we show that, at physiological concentrations, aggregation kinetics of insect cell-derived full-length wild-type p53 and p53R248Q are determined by a nucleation-growth model, rather than formation of aggregation-prone monomeric species. Self-seeding, but not cross-seeding, increases aggregation rate, confirming the aggregation process as rate determining. p53R248Q displays enhanced aggregation propensity due to decreased solubility and increased aggregation rate, forming greater numbers of larger amorphous aggregates that disrupt lipid bilayers and invokes an inflammatory response. These results suggest that p53 aggregation can occur under physiological conditions, a rate enhanced by R248Q mutation, and that aggregates formed can cause membrane damage and inflammation that may influence tumorigenesis.     

Soluble aggregates of the amyloid-beta protein activate endothelial monolayers for adhesion and subsequent transmigration of monocyte cells

Increasing evidence suggests that the deposition of amyloid plaques, composed primarily of the amyloid-β protein (Aβ), within the cerebrovasculature is a frequent occurrence in Alzheimer's disease and may play a significant role in disease progression. Accordingly, the pathogenic mechanisms by which Aβ can alter vascular function may have therapeutic implications. Despite observations that Aβ elicits a number of physiological responses in endothelial cells, ranging from alteration of protein expression to cell death, the Aβ species accountable for these responses remains unexplored. In the current study, we show that isolated soluble Aβ aggregation intermediates activate human brain microvascular endothelial cells for both adhesion and subsequent transmigration of monocyte cells in the absence of endothelial cell death and monolayer disruption. In contrast, unaggregated Aβ monomer and mature Aβ fibril fail to induce any change in endothelial adhesion or transmigration. Correlations between average Aβ aggregate size and observed increases in adhesion illustrate that smaller soluble aggregates are more potent activators of endothelium. These results support previous studies demonstrating heightened neuronal activity of soluble Aβ aggregates, including Aβ-derived diffusible ligands, oligomers, and protofibrils, and further show that soluble aggregates also selectively exhibit activity in a vascular cell model.     

The role of pH on instability and aggregation of sickle hemoglobin solutions

Understanding the physical basis of protein aggregation covers strong physical and biomedical interests. Sickle hemoglobin (HbS) is a point-mutant form of normal human adult hemoglobin (HbA). It is responsible for the first identified "molecular disease," as its propensity to aggregation is responsible for sickle cell disease. At moderately higher than physiological pH value, this propensity is inhibited: The rate of aggregate nucleation becomes exceedingly small and solubility after polymerization increases. These order-of-magnitude effects on polymer nucleation rates and concurrent relatively modest changes of solubility after polymerization are here shown to be related to both pH-induced changes of location and shape of the liquid-liquid demixing (LLD) region. This allows establishment of a self-consistent contact between the thermodynamics of the solution as such (i.e., the LLD region), the kinetics of fiber nucleation, the theory of percolation, and the thermodynamics of gelation. The observed pH-induced changes are largely attributable to strong perturbations of hydrophobic hydration configurations and related free energy by electric charges. Similar mechanisms of effective control of aggregate nucleation rates by means of agents such as cosolutes, pH, salts, and additives, shifting the LLD and associated regions of anomalous fluctuations, promise to be relevant to the whole field of protein aggregation pathologies.     

Divergent genetic control of protein solubility and conformational quality in Escherichia coli

In bacteria, protein overproduction results in the formation of inclusion bodies, sized protein aggregates showing amyloid-like properties such as seeding-driven formation, amyloid-tropic dye binding, intermolecular β-sheet architecture and cytotoxicity on mammalian cells. During protein deposition, exposed hydrophobic patches force intermolecular clustering and aggregation but these aggregation determinants coexist with properly folded stretches, exhibiting native-like secondary structure. Several reports indicate that inclusion bodies formed by different enzymes or fluorescent proteins show detectable biological activity. By using an engineered green fluorescent protein as reporter we have examined how the cell quality control distributes such active but misfolded protein species between the soluble and insoluble cell fractions and how aggregation determinants act in cells deficient in quality control functions. Most of the tested genetic deficiencies in different cytosolic chaperones and proteases (affecting DnaK, GroEL, GroES, ClpB, ClpP and Lon at different extents) resulted in much less soluble but unexpectedly more fluorescent polypeptides. The enrichment of aggregates with fluorescent species results from a dramatic inhibition of ClpP and Lon-mediated, DnaK-surveyed green fluorescent protein degradation, and it does not perturb the amyloid-like architecture of inclusion bodies. Therefore, the Escherichia coli quality control system promotes protein solubility instead of conformational quality through an overcommitted proteolysis of aggregation-prone polypeptides, irrespective of their global conformational status and biological properties.     

Aggregated proteins accelerate but do not increase the aggregation of D-glyceraldehyde-3-phosphate dehydrogenase. Specificity of protein aggregation

The effect of protein aggregates on the aggregation of d-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) during unfolding and refolding has been studied. The aggregation of GAPDH follows a sigmoid course. The presence of protein aggregates increases the aggregation rate during unfolding and refolding of GAPDH but does not change the extent of aggregation and the final renaturation yield. It is suggested that protein aggregates function as seeds for aggregation via hydrophobic interaction with only GAPDH folding intermediates destined to aggregate and do not affect the distribution between pathways leading to correct folding and aggregation. Moreover, two different proteins do not interfere with each other during their simultaneous refolding together in a buffer. These findings provide insight into a mechanism by which cells prevent protein folding against the interference from aggregation of other proteins.     

Production of soluble ScFvs with C-terminal-free thiol for site-specific conjugation or stable dimeric ScFvs on demand

ScFv recombinant antibody fragments can provide specific tumor binding modules for targeting drugs. In the process of building multimeric tumor targeting pharmaceuticals, a prerequisite is the conservation of functional scFv antigen binding domains, thereby excluding scFv random conjugation to a carrier molecule or to another scFv. The pCANTAB 5E phage display/expression vector was genetically engineered to express any scFv gene as scFv with an additional C-terminal cysteine (scFv-Cys) such that the specific conjugation site is removed from the binding domain. Selected scFvs derived from an anti-MUC-1 scFv phage library were expressed in pCANTAB 5E and its modified version pCANTAB 5E Cys vectors, and compared for key characteristics. Production yields of scFv and scFv-Cys in shaker flask and biofermentor were compared. In the absence of a reducing agent, stable dimers (covalent scFv homodimers (scFv-Cys)2) were the major form of scFv-Cys. These diabodies provided substantial signal enhancement for immunohistochemical staining of tissues. In the presence of a reducing agent, scFv-Cys molecules remained monomeric, with the free SH available for conjugation to a PEG(maleimide)2 scaffold to form immunoreactive PEG(scFv)2 bioconjugates. ScFv expression from pCANTAB 5E Cys allowed for the production of soluble scFv-Cys protein from E.coli, either as stable scFv-Cys or (scFv-Cys)2. ScFv-Cys can be used for conjugation to PEG to form bivalent PEG (scFv-Cys)2 molecules or used as (scFv-Cys)2 for increased sensitivity in IHC.