Galectin-1 as a fusion partner for the production of soluble and folded human β-1,4-galactosyltransferase-T7 in E. coli

The expression of recombinant proteins in Escherichia coli often leads to inactive aggregated proteins known as the inclusion bodies. To date, the best available tool has been the use of fusion tags, including the carbohydrate-binding protein; e.g., the maltose-binding protein (MBP) that enhances the solubility of recombinant proteins. However, none of these fusion tags work universally with every partner protein. We hypothesized that galectins, which are also carbohydrate-binding proteins, may help as fusion partners in folding the mammalian proteins in E. coli. Here we show for the first time that a small soluble lectin, human galectin-1, one member of a large galectin family, can function as a fusion partner to produce soluble folded recombinant human glycosyltransferase, β-1,4-galactosyltransferase-7 (β4Gal-T7), in E. coli. The enzyme β4Gal-T7 transfers galactose to xylose during the synthesis of the tetrasaccharide linker sequence attached to a Ser residue of proteoglycans. Without a fusion partner, β4Gal-T7 is expressed in E. coli as inclusion bodies. We have designed a new vector construct, pLgals1, from pET-23a that includes the sequence for human galectin-1, followed by the Tev protease cleavage site, a 6× His-coding sequence, and a multi-cloning site where a cloned gene is inserted. After lactose affinity column purification of galectin-1-β4Gal-T7 fusion protein, the unique protease cleavage site allows the protein β4Gal-T7 to be cleaved from galectin-1 that binds and elutes from UDP-agarose column. The eluted protein is enzymatically active, and shows CD spectra comparable to the folded β4Gal-T1. The engineered galectin-1 vector could prove to be a valuable tool for expressing other proteins in E. coli.     

Definitive evidence that a single N-glycan among three glycans on inducible costimulator is required for proper protein trafficking and ligand binding

Glycosylation is a widespread post-translational modification found in glycoproteins. Glycans play key roles in protein folding, quality control in the endoplasmic reticulum (ER) and protein trafficking within cells. However, it remains unclear whether all positions of protein glycosylation are involved in glycan functions, or if specific positions have individual roles. Here we demonstrate the integral involvement of a specific N-glycan from amongst the three glycans present on inducible costimulator (ICOS), a T-cell costimulatory molecule, in proper protein folding and intracellular trafficking to the cell surface membrane. We found that glycosylation-defective mutant proteins lacking N-glycan at amino-acid position 89 (N89), but not proteins lacking either N23 or N110, were retained within the cell and were not detected on the cell surface membrane. Additional evidence suggested that N89 glycosylation was indirectly involved in ICOS ligand binding. These data suggest that amongst the three putative ICOS glycosylation sites, N89 is required for proper ICOS protein folding in the ER, intracellular trafficking and ligand binding activity. This study represents a substantial contribution to the current mechanistic understanding of the necessity and potential functions of a specific N-glycan among the multiple glycans of glycoproteins.     

A novel Escherichia coli solubility enhancer protein for fusion expression of aggregation-prone heterologous proteins

Through the proteome analysis of Escherichia coli BL21(DE3), we previously identified the stress-responsive protein, arsenate reductase (ArsC), that showed a high cytoplasmic solubility and a folding capacity even in the presence of stress-inducing reagents. In this study, we used ArsC as an N-terminal fusion partner to synthesize nine aggregation-prone proteins as water-soluble forms. As a result, solubility of the aggregation-prone proteins increased dramatically by the fusion of ArsC, due presumably to its tendency to facilitate the folding of target proteins. Also, we evaluated and confirmed the efficacy of ArsC-fusion expression in making the fusion-expressed target proteins have their own native function or structure. That is, the self-assembly function of human ferritin light chain, l-arginine-degrading function of arginine deiminase, and the correct secondary structure of human granulocyte colony stimulating factor were clearly observed through transmission electron microscope analysis, colorimetric enzyme activity assay, and circular dichroism, respectively. It is strongly suggested that ArsC can be in general an efficient fusion expression partner for the production of soluble and active heterologous proteins in E. coli.     

The Ability to Enhance the Solubility of Its Fusion Partners Is an Intrinsic Property of Maltose-Binding Protein but Their Folding Is Either Spontaneous or Chaperone-Mediated

Escherichia coli maltose binding protein (MBP) is commonly used to promote the solubility of its fusion partners. To investigate the mechanism of solubility enhancement by MBP, we compared the properties of MBP fusion proteins refolded in vitro with those of the corresponding fusion proteins purified under native conditions. We fused five aggregation-prone passenger proteins to 3 different N-terminal tags: His₆-MBP, His₆-GST and His₆. After purifying the 15 fusion proteins under denaturing conditions and refolding them by rapid dilution, we recovered far more of the soluble MBP fusion proteins than their GST- or His-tagged counterparts. Hence, we can reproduce the solubilizing activity of MBP in a simple in vitro system, indicating that no additional factors are required to mediate this effect. We assayed both the soluble fusion proteins and their TEV protease digestion products (i.e., with the N-terminal tag removed) for biological activity. Little or no activity was detected for some fusion proteins whereas others were quite active. When the MBP fusions proteins were purified from E. coli under native conditions they were all substantially active. These results indicate that the ability of MBP to promote the solubility of its fusion partners in vitro sometimes, but not always, results in their proper folding. We show that the folding of some passenger proteins is mediated by endogenous chaperones in vivo. Hence, MBP serves as a passive participant in the folding process; passenger proteins either fold spontaneously or with the assistance of chaperones.     

Analysis of protein aggregation kinetics using short amino acid peptide tags

Understanding protein solubility, and consequently aggregation, is an important issue both from an academic and a biotechnological application viewpoints. Here we report the effects of 10 representative amino acids on the aggregation kinetics of proteins. The effects were determined by measuring the solubility of a simplified bovine pancreatic trypsin inhibitor (BPTI) variant, to which short artificial tags containing the amino acid of interest were added at its C-terminus. We determined the solubility of the tagged variants as a function of equilibration time (20 min to 48 h) and total protein concentration ranging from 0.10 mg/ml to 25.0 mg/ml. We observed, as anticipated, that proteins precipitated when the total protein concentration exceeded a critical value. However, when the total protein concentration was further increased, the apparent solubility reached a concentration above the critical value, and slowly decreased to a value under the critical concentration upon increasing the equilibration period. We rationalized these observations by identifying three different solubility values, the “transient solubility (TS)”, the “aggregation initiation concentration (AIC)” and the “long-term solubility (LS)”. AIC and LS are parameters determined essentially by the amino acid types composing the tags and could be considered as an amino acid's intrinsic property. On the other hand, TS is an apparent solubility that is measured after some (20 min in our case) equilibration time and is often considered as the “solubility” of the protein. Similar aggregation kinetic patterns were observed with natural proteins, indicating the generality of the observations made using our model protein.     

Conversion of a Chaperonin GroEL-independent Protein into an Obligate Substrate

Chaperones assist protein folding by preventing unproductive protein aggregation in the cell. In Escherichia coli, chaperonin GroEL/GroES (GroE) is the only indispensable chaperone and is absolutely required for the de novo folding of at least ∼60 proteins. We previously found that several orthologs of the obligate GroE substrates in Ureaplasma urealyticum, which lacks the groE gene in the genome, are E. coli GroE-independent folders, despite their significant sequence identities. Here, we investigated the key features that define the GroE dependence. Chimera or random mutagenesis analyses revealed that independent multiple point mutations, and even single mutations, were sufficient to confer GroE dependence on the Ureaplasma MetK. Strikingly, the GroE dependence was well correlated with the propensity to form protein aggregates during folding. The results reveal the delicate balance between GroE dependence and independence. The function of GroE to buffering the aggregation-prone mutations plays a role in maintaining higher genetic diversity of proteins.     

5S rRNA-assisted DnaK refolding

Although accumulating evidence has revealed that most proteins can fold without the assistance of molecular chaperones, little attention has been paid to other types of chaperoning macromolecules. A variety of proteins interact with diverse RNA molecules in vivo, suggesting a potential role of RNAs for folding of their interacting proteins. Here we show that the in vitro refolding of a representative molecular chaperone, DnaK, an Escherichia coli homolog of Hsp70, could be assisted by its interacting 5S rRNA. The folding enhancement occurred in RNA concentration and its size dependent manner whereas neither the RNA with the reverse sequence of 5S rRNA nor the RNase pretreated 5S rRNA stimulated the folding in vitro. Based on our results, we propose that 5S rRNA could exert the chaperoning activity on DnaK during the folding process. The results suggest an interesting possibility that the folding of RNA-interacting proteins could be assisted by their cognate RNA ligands.     

Stepwise Folding of an Autotransporter Passenger Domain Is Not Essential for Its Secretion

Autotransporters are a superfamily of virulence proteins produced by Gram-negative bacteria. They consist of an N-terminal β-helical domain (“passenger domain”) that is secreted into the extracellular space and a C-terminal β-barrel domain (“β-domain”) that anchors the protein to the outer membrane. Because the periplasm lacks ATP, vectorial folding of the passenger domain in a C-to-N-terminal direction has been proposed to drive the secretion reaction. Consistent with this hypothesis, mutations that disrupt the folding of the C terminus of the passenger domain of the Escherichia coli O157:H7 autotransporter EspP have been shown to cause strong secretion defects. Here, we show that point mutations introduced at specific locations near the middle or N terminus of the EspP β-helix that perturb folding also impair secretion, but to a lesser degree. Surprisingly, we found that even multiple mutations that potentially abolish the stability of several consecutive rungs of the β-helix only moderately reduce secretion efficiency. Although these results provide evidence that the free energy derived from passenger domain folding contributes to secretion efficiency, they also suggest that a significant fraction of the energy required for secretion is derived from another source.     

Understanding protein folding from globular to amyloid state: Aggregation: Darker side of protein

Folding and unfolding are crucial ways of modulating biological activity and targeting proteins to different cellular locations. In the living system, protein folding occurs in a very crowded environment, often assisted with helper proteins. In some cases this pathway can go off beam and the protein can either misfold or aggregate or form structures of elongated-unbranched morphology known as amyloid fibrils. Protein folding is not just an academic matter. Recombinant biotechnology and pharmaceutical industries are some of the fields where both theoretical and practical knowledge of protein folding is required. Misfolded protein and amyloid fibrils that escape the cellular quality control check are the basic reason of a number of increasingly widespread neurodegenerative diseases such as Alzheimer's and variant Creutzfeldt-Jakob etc. Thus, protein folding study also emerges as an interesting area in the field of biomedical research. This review deals with basic concepts related to protein folding and misfolding forming toxic aggregates and amyloid fibrils as well as disease associated with them. A more practical approach will be revealed to the early diagnosis of aggregation-prone diseases and amyloid states and their balanced therapeutics.     

Chaperones GroEL/GroES Accelerate the Refolding of a Multidomain Protein through Modulating On-pathway Intermediates

Despite a vast amount information on the interplay of GroEL, GroES, and ATP in chaperone-assisted folding, the molecular details on the conformational dynamics of folding polypeptide during its GroEL/GroES-assisted folding cycle is quite limited. Practically no such studies have been reported to date on large proteins, which often have difficulty folding in vitro. The effect of the GroEL/GroES chaperonin system on the folding pathway of an 82-kDa slow folding protein, malate synthase G (MSG), was investigated. GroEL bound to the burst phase intermediate of MSG and accelerated the slowest kinetic phase associated with the formation of native topology in the spontaneous folding pathway. GroEL slowly induced conformational changes on the bound burst phase intermediate, which was then transformed into a more folding-compatible form. Subsequent addition of ATP or GroES/ATP to the GroEL-MSG complex led to the formation of the native state via a compact intermediate with the rate several times faster than that of spontaneous refolding. The presence of GroES doubled the ATP-dependent reactivation rate of bound MSG by preventing multiple cycles of its GroEL binding and release. Because GroES bound to the trans side of GroEL-MSG complex, it may be anticipated that confinement of the substrate underneath the co-chaperone is not required for accelerating the rate in the assisted folding pathway. The potential role of GroEL/GroES in assisted folding is most likely to modulate the conformation of MSG intermediates that can fold faster and thereby eliminate the possibility of partial aggregation caused by the slow folding intermediates during its spontaneous refolding pathway.     

Protein aggregation during overexpression limited by peptide extensions with large net negative charge

Folding of the human coxsackie and adenovirus receptor immunoglobulin (Ig) variable-type domain (CAR D1) during overexpression in the Escherichia coli cytoplasm was shown previously to be partially rescued by fusion to a 22-residue C-terminal peptide. Here, peptide sequence features required for solubilization and folding of CAR D1 and similar Ig variable-type domains from two other human membrane proteins were investigated. Peptide extensions with net negative charge > -6 fully solubilized CAR D1, and approximately half of the peptide-solubilized protein was correctly folded. The Ig variable-type domains from human A33 antigen and myelin P-zero proteins were only partially solubilized by peptide extensions with net charge of -12, however, and only the solubilized P-zero domain appeared to fold correctly whereas the A33 domain formed soluble microaggregates of misfolded protein. Our results suggest a model where the large net charge of peptide extensions increases electrostatic repulsion between nascent polypeptides. The resulting decrease in aggregation rate can enable some polypeptides to fold spontaneously into their native protein conformations. Analysis of the solubility and folding status of sets of structurally homologous proteins, such as the Ig variable-type domains described here, during overexpression could provide insights into how amino acid and gene sequences influence the efficiency of spontaneous protein folding.     

Protein folding in confined and crowded environments

Confinement and crowding are two major factors that can potentially impact protein folding in cellular environments. Theories based on considerations of excluded volumes predict disparate effects on protein folding stability for confinement and crowding: confinement can stabilize proteins by over 10k_BT but crowding has a very modest effect on stability. On the other hand, confinement and crowding are both predicted to favor conformations of the unfolded state which are compact, and consequently may increase the folding rate. These predictions are largely borne out by experimental studies of protein folding under confined and crowded conditions in the test tube. Protein folding in cellular environments is further complicated by interactions with surrounding surfaces and other factors. Concerted theoretical modeling and test-tube and in vivo experiments promise to elucidate the complexity of protein folding in cellular environments.     

Identical RNA-Protein Interactions in Vivo and in Vitro and a Scheme of Folding the Newly Synthesized Proteins by Ribosomes

A distinct three-dimensional shape of rRNA inside the ribosome is required for the peptidyl transfer activity of its peptidyltransferase center (PTC). In contrast, even the in vitro transcribed PTC RNA interacts with unfolded protein(s) at about five sites to let them attain their native states. We found that the same set of conserved nucleotides in the PTC interact identically with nascent and chemically unfolded proteins in vivo and in vitro, respectively. The time course of this interaction, difficult to follow in vivo, was observed in vitro. It suggested nucleation of folding of cytosolic globular proteins vectorially from hydrophilic N to hydrophobic C termini, consistent with our discovery of a regular arrangement of cumulative hydrophobic indices of the peptide segments of cytosolic proteins from N to C termini. Based on this observation, we propose a model here for the nucleation of folding of the nascent protein chain by the PTC.     

Predicting protein folding cores by empirical potential functions

Theoretical and in vitro experiments suggest that protein folding cores form early in the process of folding, and that proteins may have evolved to optimize both folding speed and native-state stability. In our previous work (Chen et al., Structure, 14 (2006) 1401), we developed a set of empirical potential functions and used them to analyze interaction energies among secondary-structure elements in two β-sandwich proteins. Our work on this group of proteins demonstrated that the predicted folding core also harbors residues that form native-like interactions early in the folding reaction. In the current work, we have tested our empirical potential functions on structurally-different proteins for which the folding cores have been revealed by protein hydrogen-deuterium exchange experiments. Using a set of 29 unrelated proteins, which have been extensively studied in the literature, we demonstrate that the average prediction result from our method is significantly better than predictions based on other computational methods. Our study is an important step towards the ultimate goal of understanding the correlation between folding cores and native structures.     

Novel Thioredoxin-related Transmembrane Protein TMX4 Has Reductase Activity

In the endoplasmic reticulum (ER), a number of thioredoxin (Trx) superfamily proteins are present to enable correct disulfide bond formation of secretory and membrane proteins via Trx-like domains. Here, we identified a novel transmembrane Trx-like protein 4 (TMX4), in the ER of mammalian cells. TMX4, a type I transmembrane protein, was localized to the ER and possessed a Trx-like domain that faced the ER lumen. A maleimide alkylation assay showed that a catalytic CXXC motif in the TMX4 Trx-like domain underwent changes in its redox state depending on cellular redox conditions, and, in the normal state, most of the endogenous TMX4 existed in the oxidized form. Using a purified recombinant protein containing the Trx-like domain of TMX4 (TMX4-Trx), we confirmed that this domain had reductase activity in vitro. The redox potential of this domain (−171.5 mV; 30 °C at pH 7.0) indicated that TMX4 could work as a reductase in the environment of the ER. TMX4 had no effect on the acceleration of ER-associated degradation. Because TMX4 interacted with calnexin and ERp57 by co-immunoprecipitation assay, the role of TMX4 may be to enable protein folding in cooperation with these proteins consisting of folding complex in the ER.     

Structure-function analysis of the C-terminal domain of CNM67, a core component of the Saccharomyces cerevisiae spindle pole body

The spindle pole body of the budding yeast Saccharomyces cerevisiae has served as a model system for understanding microtubule organizing centers, yet very little is known about the molecular structure of its components. We report here the structure of the C-terminal domain of the core component Cnm67 at 2.3 Å resolution. The structure determination was aided by a novel approach to crystallization of proteins containing coiled-coils that utilizes globular domains to stabilize the coiled-coils. This enhances their solubility in Escherichia coli and improves their crystallization. The Cnm67 C-terminal domain (residues Asn-429—Lys-581) exhibits a previously unseen dimeric, interdigitated, all α-helical fold. In vivo studies demonstrate that this domain alone is able to localize to the spindle pole body. In addition, the structure reveals a large functionally indispensable positively charged surface patch that is implicated in spindle pole body localization. Finally, the C-terminal eight residues are disordered but are critical for protein folding and structural stability.     

Sequential folding of transfer RNA: A nuclear magnetic resonance study of successively longer tRNA fragments with a common 5′ end

Most folding studies on proteins and nucleic acids have been addressed to the transition between the folded and unfolded states of an intact molecule, where an entire residue sequence is present during the folding event. However, since these polymers are synthesized sequentially from one terminus to the other in vivo, their folding pathways may be influenced greatly by the sequential appearance of the residues as a function of time.The three-dimensional structure of yeast tRNA^Phe in the crystalline state is correlated with 360 MHz proton nuclear magnetic resonances from three fragments plus an intact molecule of the tRNA that share a common 5′ end and are in a solution condition similar to that of the crystal structure. This has allowed identification of folded structures present in the fragments and presumably present in the growing tRNA molecule as it is being synthesized from the 5′ end. The experiments show that only the correct stems are formed in the fragments; no additional or competing helical region is produced. This suggests that in the biosynthesis of this tRNA, correct folding of helical stems occurs before the entire molecule is formed. Further, some of the tertiary interactions (hydrogen bonds) found in the crystal structure are also probably present before the synthesis is completed. These findings are generalized to consider the precursor of the tRNA as well as other tRNAs.     

Backbones of Folded Proteins Reveal Novel Invariant Amino Acid Neighborhoods

Abstract

Folding of naturally occurring proteins has eluded a universal molecular level explanation till date. Rather, there is an abundance of diverse views on dominant factors governing protein folding. Through rigorous analyses of several thousand crystal structures, we observe that backbones of folded proteins display some remarkable invariant features. Folded proteins are characterized by spatially well-defined, distance dependent, and universal, neighborhoods of amino acids which defy any of the conventionally prevalent views. These findings present a compelling case for a newer view of protein folding which takes into account solvent mediated and amino acid shape and size assisted optimization of the tertiary structure of the polypeptide chain to make a functional protein.