Co-translational folding of an eukaryotic multidomain protein in a prokaryotic translation system

Continuous monitoring of the enzymatic activity of newly synthesized firefly luciferase in Escherichia coli cell-free translation system was performed to record folding kinetics of this multidomain eukaryotic protein in the prokaryotic cytosol. Whereas in vitro refolding of denatured luciferase in prokaryotic cytosol occurred with a low yield of active enzyme and took about an hour, the enzyme acquired its native structure immediately upon release from the ribosome, as seen from the immediate halt of active luciferase accumulation upon blocking of translation with inhibitors. The nascent luciferase was also capable of acquiring the active conformation prior to release from the ribosome, when its C terminus was extended with a polypeptide segment. Specific enzymatic activity of the firefly luciferase was found to be equally high irrespective of whether this protein was synthesized in eukaryotic or prokaryotic translation systems. The data presented demonstrate the fundamental ability of prokaryotic cytosol to support effective co-translational protein folding in general and co-translational folding of multidomain proteins in particular.     

Function of trigger factor and DnaK in multidomain protein folding: increase in yield at the expense of folding speed

Trigger factor and DnaK protect nascent protein chains from misfolding and aggregation in the E. coli cytosol, but how these chaperones affect the mechanism of de novo protein folding is not yet understood. Upon expression under chaperone-depleted conditions, multidomain proteins such as bacterial beta-galactosidase (beta-gal) and eukaryotic luciferase fold by a rapid but inefficient default pathway, tightly coupled to translation. Trigger factor and DnaK improve the folding yield of these proteins but markedly delay the folding process both in vivo and in vitro. This effect requires the dynamic recruitment of additional trigger factor molecules to translating ribosomes. While beta-galactosidase uses this chaperone mechanism effectively, luciferase folding in E. coli remains inefficient. The efficient cotranslational domain folding of luciferase observed in the eukaryotic system is not compatible with the bacterial chaperone system. These findings suggest important differences in the coupling of translation and folding between bacterial and eukaryotic cells.     

Comparative expression of wild-type and highly soluble mutant His103Leu of hydroxynitrile lyase from Manihot esculenta in prokaryotic and eukaryotic expression systems

Low protein solubility and inclusion body formation represent big challenges in production of recombinant proteins in Escherichia coli. We have recently reported functional expression of hydroxynitrile lyase from Manihot esculenta, MeHNL, in E. coli with high in vivo solubility and activity using directed evolution. As a part of attempts to clarify the mechanism of this phenomenon, we have described the possibility of expression of the highly active and soluble mutant MeHNL-His103Leu as well as wild-type enzyme in several expression systems. Methylotrophic yeast Pichia pastoris, protozoan host Leishmania tarentolae and two cell-free translations, including an E. coli lysate (WakoPURE system) and wheat germ translation system were used to compare expression profiles of the genes. Two distinguishable protein expression patterns were observed in prokaryotic and eukaryotic-based systems. The wild-type and mutant enzyme showed high activity for both genes (up to 10 U/ml) in eukaryotic hosts P. pastoris and L. tarentolae, while those of E. coli exhibited about 1 and 15 U/ml, respectively. The different activity level in prokaryotic systems but the same level among the eukaryotic hosts indicate the phenomenon is specific to the E. coli system. Both the wild-type and mutant enzymes were functionally expressed in eukaryotic systems, probably using the folding assistants such as chaperones. Properties of expression systems used in this study were precisely compared, too.     

Synthesis and site-directed fluorescence labeling of azido proteins using eukaryotic cell-free orthogonal translation systems

Eukaryotic cell-free systems based on wheat germ and Spodoptera frugiperda insect cells were equipped with an orthogonal amber suppressor tRNA–synthetase pair to synthesize proteins with a site-specifically incorporated p-azido-l-phenylalanine residue in order to provide their chemoselective fluorescence labeling with azide-reactive dyes by Staudinger ligation. The specificity of incorporation and bioorthogonality of labeling within complex reaction mixtures was shown by means of translation and fluorescence detection of two model proteins: β-glucuronidase and erythropoietin. The latter contained the azido amino acid in proximity to a signal peptide for membrane translocation into endogenous microsomal vesicles of the insect cell-based system. The results indicate a stoichiometric incorporation of the azido amino acid at the desired position within the proteins. Moreover, the compatibility of cotranslational protein translocation, including glycosylation and amber suppression-based incorporation of p-azido-l-phenylalanine within a cell-free system, is demonstrated. The presented approach should be particularly useful for providing eukaryotic and membrane-associated proteins for investigation by fluorescence-based techniques.     

The folding and evolution of multidomain proteins

Analyses of genomes show that more than 70% of eukaryotic proteins are composed of multiple domains. However, most studies of protein folding focus on individual domains and do not consider how interactions between domains might affect folding. Here, we address this by analysing the three-dimensional structures of multidomain proteins that have been characterized experimentally and observe that where the interface is small and loosely packed, or unstructured, the folding of the domains is independent. Furthermore, recent studies indicate that multidomain proteins have evolved mechanisms to minimize the problems of interdomain misfolding.     

Trigger factor lacking the PPIase domain can enhance the folding of eukaryotic multi-domain proteins in Escherichia coli

Recombinant expression of eukaryotic proteins in bacteria often results in misfolding and aggregation. The ribosome-binding Trigger factor (TF) is the first molecular chaperone that interacts with nascent polypeptide chains in bacteria. Here we show that mutant TF lacking the PPIase domain (TFNC) is more efficient than wild-type TF in enhancing the folding yield of multi-domain proteins such as firefly luciferase. We find that TFNC has a shorter residence time on nascent chains, thus facilitating co-translational folding. By delaying folding relative to translation, the PPIase domain may increase the propensity of misfolding for certain eukaryotic proteins that rely on a mechanism of co-translational, domain-wise folding.     

A low-Mr factor isolated from Escherichia coli inhibits eukaryotic in vitro protein synthesis

The effect of a low-Mr factor, partially purified from E. coli B, was investigated in E. coli, reticulocyte, and wheat germ lysate in vitro protein synthesis systems. Equal concentrations of factor were needed to inhibit protein synthesis in the eukaryotic system as compared to the prokaryotic system. Experiments suggested that the factor inhibits the initiation step in the eukaryotic systems.     

5'-poly(A) sequence as an effective leader for translation in eukaryotic cell-free systems

Poly(A) sequence of 25 adenylic residues placed immediately before the start codons of the green fluorescent protein (GFP) and firefly luciferase (Luc) mRNAs is shown to provide a high rate of translation of the heterologous messages in eukaryotic cell-free translation systems. Also the poly(A) leader is found to provide the abolition of the inhibition of translation at excess mRNA concentrations. The possibility of the practical use of the constructs with the poly(A) leader for preparative protein production is demonstrated in the wheat germ continuous-exchange cell-free (CECF) translation system.     

A system using convertible vectors for screening soluble recombinant proteins produced in Escherichia coli from randomly fragmented cDNAs

Protein insolubility is a major problem when producing recombinant proteins (e.g., to be used as antigens) from large cDNAs in Escherichia coli. Here, we describe a system using three convertible plasmid vectors to screen for soluble proteins produced in E. coli. This system experimentally identified any random cDNA fragments producing soluble protein domains. Shotgun fragments introduced into any of our three plasmids, which contain Gateway recombination sites, fused in-frame to the ORF of the protein tag. These plasmids produced N-terminal GST- and C-terminal three-frame-adaptive FLAG-tagged proteins, kanamycin-resistant gene-tagged proteins (which were pre-selected for in-frame fused cDNAs), or GFP-tagged fusion proteins. The latter is useful as a fluorescence indicator of protein folding. The Gateway recombination sites promote smooth conversion for enrichment of in-frame clones and facilitate both protein solubility assays and final production of proteins without the C-terminal tag. This high-throughput screening method is particularly useful for procedures that require the handling of many cDNAs in parallel.     

Different mechanistic requirements for prokaryotic and eukaryotic chaperonins: a lattice study

MOTIVATION: The folding of many proteins in vivo and in vitro is assisted by molecular chaperones. A well-characterized molecular chaperone system is the chaperonin GroEL/GroES from Escherichia coli which has a homolog found in the eukaryotic cytosol called CCT. All chaperonins have a ring structure with a cavity in which the substrate protein folds. An interesting difference between prokaryotic and eukaryotic chaperonins is in the nature of the ATP-mediated conformational changes that their ring structures undergo during their reaction cycle. Prokaryotic chaperonins are known to exhibit a highly cooperative concerted change of their cavity surface while in eukaryotic chaperonins the change is sequential. Approximately 70% of proteins in eukaryotic cells are multi-domain whereas in prokaryotes single-domain proteins are more common. Thus, it was suggested that the different modes of action of prokaryotic and eukaryotic chaperonins can be explained by the need of eukaryotic chaperonins to facilitate folding of multi-domain proteins. RESULTS: Using a 2D square lattice model, we generated two large populations of single-domain and double-domain substrate proteins. Chaperonins were modeled as static structures with a cavity wall with which the substrate protein interacts. We simulated both concerted and sequential changes of the cavity surfaces and demonstrated that folding of single-domain proteins benefits from concerted but not sequential changes whereas double-domain proteins benefit also from sequential changes. Thus, our results support the suggestion that the different modes of allosteric switching of prokaryotic and eukaryotic chaperonin rings have functional implications as it enables eukaryotic chaperonins to better assist multi-domain protein folding.     

Cotranslational formation of active photoprotein obelin in a cell-free translation system: direct ultrahigh sensitive measure of the translation course

Translation of apoobelin mRNA in a cell-free wheat germ translation system in the presence of coelenterazine and molecular oxygen results in cotranslational formation of active photoprotein. Active obelin formation is recorded by its luminescence, either direct in the translation mixture in the presence of coelenterazine and calcium ions or in aliquots from the translation mixture. In the second case translation is carried out with coelenterazine and EGTA. Registration of the translation course by luminescence of the synthesized product in both cases allows use of apoobelin mRNA at very low concentrations as an internal marker for immediate measure of protein biosynthesis activity of in vitro translation systems. It is shown that the simultaneous translation of any other mRNA does not affect translation of photoprotein mRNAs under standard conditions. Continuous registration of luminescence in a cuvette of a liquid scintillation counter in photon-counting mode varies the time of signal accumulation in a wide temporal range, thus increasing the numerical values of the recorded signals. Registration of photoprotein luminescence during translation can be used to obtain additional information about the translation process, for example codon reading speed, about protein folding, and about the formation of active proteins on ribosomes.     

Consequences of the overexpression of a eukaryotic membrane protein, the human KDEL receptor, in Escherichia coli

Escherichia coli is the most widely used host for producing membrane proteins. Thus far, to study the consequences of membrane protein overexpression in E. coli, we have focussed on prokaryotic membrane proteins as overexpression targets. Their overexpression results in the saturation of the Sec translocon, which is a protein-conducting channel in the cytoplasmic membrane that mediates both protein translocation and insertion. Saturation of the Sec translocon leads to (i) protein misfolding/aggregation in the cytoplasm, (ii) impaired respiration, and (iii) activation of the Arc response, which leads to inefficient ATP production and the formation of acetate. The overexpression yields of eukaryotic membrane proteins in E. coli are usually much lower than those of prokaryotic ones. This may be due to differences between the consequences of the overexpression of prokaryotic and eukaryotic membrane proteins in E. coli. Therefore, we have now also studied in detail how the overexpression of a eukaryotic membrane protein, the human KDEL receptor, affects E. coli. Surprisingly, the consequences of the overexpression of a prokaryotic and a eukaryotic membrane protein are very similar. Strain engineering and likely also protein engineering can be used to remedy the saturation of the Sec translocon upon overexpression of both prokaryotic and eukaryotic membrane proteins in E. coli.     

A set of dual promoter vectors for high throughput cloning, screening, and protein expression in eukaryotic and prokaryotic systems from a single plasmid

ABSTRACT: BACKGROUND: The ability to produce the same recombinant protein in both prokaryotic and eukaryotic cells offers many experimental opportunities. However, the cloning of the same gene into multiple plasmids is required, which is time consuming, laborious and still may not produce soluble, stable protein in sufficient quantities. We have developed a set of expression vectors that allows for ligation-independent cloning and rapid functional screening for protein expression in both E. coli and S. cerevisiae. RESULTS: A set of expression vectors was made that can express the same open reading frame in E. coli (via the T7 phage promoter) and in S. cerevisiae (via the CUP1 or MET25 promoter). These plasmids also contain the essential elements for replication and selection in both cell types and have several advantages: they allow for cloning of genes by homologous recombination in yeast, protein expression can be determined before plasmid isolation and sequencing, and a GST-fusion tag is added to aid in soluble expression and purification. We have also included a TEV recognition site that allows for the specific cleavage of the fusion proteins to yield native proteins. CONCLUSIONS: The dual promoter vectors can be used for rapid cloning, expression, and purification of target proteins from both prokaryotic and eukaryotic systems with the ability to study post-translation modifications.     

A versatile selection system for folding competent proteins using genetic complementation in a eukaryotic host

Recombinant expression of native or modified eukaryotic proteins is pivotal for structural and functional studies and for industrial and pharmaceutical production of proteins. However, it is often impeded by the lack of proper folding. Here, we present a stringent and broadly applicable eukaryotic in vivo selection system for folded proteins. It is based on genetic complementation of the Schizosaccharomyces pombe growth marker gene invertase fused C‐terminally to a protein library. The fusion proteins are directed to the secretion system, utilizing the ability of the eukaryotic protein quality‐control systems to retain misfolded proteins in the ER and redirect them for cytosolic degradation, thereby only allowing folded proteins to reach the cell surface. Accordingly, the folding potential of the tested protein determines the ability of autotrophic colony growth. This system was successfully demonstrated using a complex insertion mutant library of TNF‐α, from which different folding competent mutant proteins were uncovered.     

Cotranslational Folding of a Pentarepeat β-Helix Protein

It is becoming increasingly clear that many proteins start to fold cotranslationally before the entire polypeptide chain has been synthesized on the ribosome. One class of proteins that a priori would seem particularly prone to cotranslational folding is repeat proteins, that is, proteins that are built from an array of nearly identical sequence repeats. However, while the folding of repeat proteins has been studied extensively in vitro with purified proteins, only a handful of studies have addressed the issue of cotranslational folding of repeat proteins. Here, we have determined the structure and studied the cotranslational folding of a β-helix pentarepeat protein from the human pathogen Clostridium botulinum—a homolog of the fluoroquinolone resistance protein MfpA—using an assay in which the SecM translational arrest peptide serves as a force sensor to detect folding events. We find that cotranslational folding of a segment corresponding to the first four of the eight β-helix coils in the protein produces enough force to release ribosome stalling and that folding starts when this unit is ~ 35 residues away from the P-site, near the distal end of the ribosome exit tunnel. An additional folding transition is seen when the whole PENT moiety emerges from the exit tunnel. The early cotranslational formation of a folded unit may be important to avoid misfolding events in vivo and may reflect the minimal size of a stable β-helix since it is structurally homologous to the smallest known β-helix protein, a four-coil protein that is stable in solution.     

Comparison of folding rates of homologous prokaryotic and eukaryotic proteins

The rate of polypeptide chain elongation is up to one order of magnitude faster in prokaryotic cells than in eukaryotes. Here we report that the rates of in vitro refolding of orthologous prokaryotic and eukaryotic proteins correlate with their differential rates of biosynthesis. The mitochondrial and cytosolic aspartate aminotransferases of chicken and aspartate aminotransferase of Escherichia coli show pairwise sequence identities of 41-48% and nearly identical three-dimensional structures. Nevertheless, the prokaryotic enzyme refolded 6 times faster (at 25 degrees C) than the eukaryotic isoenzymes after denaturation in 6 m guanidine hydrochloride. Prokaryotic malate dehydrogenase and lactate dehydrogenase also renatured faster than their orthologous eukaryotic counterparts, suggesting that evolutionary pressure has adapted the rate of folding to the rate of elongation of polypeptide chains.     

Homologs of eukaryotic Ras superfamily proteins in prokaryotes and their novel phylogenetic correlation with their eukaryotic analogs

Ras superfamily proteins are key regulators in a wide variety of cellular processes. Previously, they were considered to be specific to eukaryotes, and MglA, a group of obviously different prokaryotic proteins, were recognized as their only prokaryotic analogs or even ancestors. Here, taking advantage of quite a current accumulation of prokaryotic genomic databases, we have investigated the existence and taxonomic distribution of Ras superfamily protein homologs in a much wider prokaryotic range, and analyzed their phylogenetic correlation with their eukaryotic analogs. Thirteen unambiguous prokaryotic homologs, which possess the GDP/GTP-binding domain with all the five characteristic motifs of their eukaryotic analogs, were identified in 12 eubacteria and one archaebacterium, respectively. In some other archaebacteria, including four methanogenic archaebacteria and three Thermoplasmales, homologs were also found, but with the GDP/GTP-binding domains not containing all the five characteristic motifs. Many more MglA orthologs were identified than in previous studies mainly in delta-proteobacteria, and all were shown to have common unique features distinct from the Ras superfamily proteins. Our phylogenetic analysis indicated eukaryotic Rab, Ran, Ras, and Rho families have the closest phylogenetic correlation with the 13 unambiguous prokaryotic homologs, whereas the other three eukaryotic protein families (SRbeta, Sar1, and Arf) branch separately from them, but have a relatively close relationship with the methanogenic archaebacterial homologs and MglA. Although homologs were identified in a relative minority of prokaryotes with genomic databases, their presence in a relatively wide variety of lineages, their unique sequence characters distinct from those of eukaryotic analogs, and the topology of our phylogenetic tree altogether do not support their origin from eukaryotes as a result of lateral gene transfer. Therefore, we argue that Ras superfamily proteins might have already emerged at least in some prokaryotic lineages, and that the seven eukaryotic protein families of the Ras superfamily may have two independent prokaryotic origins, probably reflecting the 'fusion' evolutionary history of the eukaryotic cell.