Molecular Chaperonin Research Articles

Chaperonin co-expression and chemical modification enables production of active microbial transglutaminase from E. coli cytoplasm

Microbial transglutaminase (MTG) is a usable enzyme for biomacromolecule modification. In the present study, a “molecular chaperonin” strategy was developed to produce MTG in E. coli cytoplasm with high expression level and a “small molecule-mediated chemical modification” strategy was adopted to strip propeptide chaperonin efficiently during purification. Propeptide (Pro) was expressed separately as a chaperonin to facilitate MTG expression in E. coli cytoplasm with a yield up to 300 mg or about 9 kU from 1 L fed-batch culture. Furthermore, small molecular chemicals were applied to interfere the interaction between MTG and Pro. Chemical acetylation was identified as a suitable method to strip Pro resulting in pure MTG with high specific activity up to 49.6 U/mg. The purified acetylated MTG was characterized by MS analysis. The deconvoluted mass and Peptide Sequence Tags analysis confirmed acetylation on amino groups of MTG protein. Finally, the applications of obtained MTG were demonstrated via protein polymerization of bovine serum albumin and PEGylation of human interferon-α2b. Our method provides MTG with high purity and specific activity as well as unique merit with masked amino groups thus avoiding self-polymerization and cross-linking between MTG and substrates.

Interaction with the CCT chaperonin complex limits APOBEC3A cytidine deaminase cytotoxicity.

The APOBEC3 cytidine deaminases are implicated as the cause of a prevalent somatic mutation pattern found in cancer genomes. The APOBEC3 enzymes act as viral restriction factors by mutating viral genomes. Mutation of the cellular genome is presumed to be an off-target activity of the enzymes, although the regulatory measures for APOBEC3 expression and activity remain undefined. It is therefore difficult to predict circumstances that enable APOBEC3 interaction with cellular DNA that leads to mutagenesis. The APOBEC3A (A3A) enzyme is the most potent deaminase of the family. Using proteomics, we evaluate protein interactors of A3A to identify potential regulators. We find that A3A interacts with the chaperonin-containing TCP-1 (CCT) complex, a cellular machine that assists in protein folding and function. Importantly, depletion of CCT results in A3A-induced DNA damage and cytotoxicity. Evaluation of cancer genomes demonstrates an enrichment of A3A mutational signatures in cancers with silencing mutations in CCT subunit genes. Together, these data suggest that the CCT complex interacts with A3A, and that disruption of CCT function results in increased A3A mutational activity.

First Report of Lychnis flos-cuculi as a New Host Plant for ‘Candidatus Phytoplasma asteris’ (Subgroup 16SrI-B)

Lychnis flos-cuculi L. (Caryophyllaceae) is a widespread perennial herb occurring in wet grassland habitats throughout Europe (Galeuchet et al. 2005). A double-flowered cultivar, Petite Jenny, is now common as an ornamental in gardens throughout the world. In September 2018, approximately 46% of L. flos-cuculi ‘Petite Jenny’ exhibited symptoms of phytoplasma infection in a commercial nursery in central Hungary. The affected plants did not produce flowers, and their shoot tips and surrounding leaves showed symptoms of yellowing and a bushy appearance. Total DNA was extracted from stem phloem scrapings and whole leaves of five healthy and 20 diseased plants using a phytoplasma enrichment procedure (Ahrens and Seemuller 1992). The DNA was used as a template in direct PCR primed by the phytoplasma-universal primers P1/P7 followed by nested PCR primed by fU5/rU3 (Lorenz et al. 1995) with Phusion High-Fidelity DNA polymerase (NEB, Ipswich, MA). Reactions containing template DNA from the symptomatic plants yielded ribosomal RNA gene amplicons of the expected size (1.8 and 0.9 kb for P1/P7 and fU5/rU3, respectively) but not from asymptomatic plants. Products of P1/P7-primed PCR were gel purified and ligated into the pJET1.2/blunt cloning vector using a CloneJET PCR cloning kit (Thermo Fisher Scientific, Waltham, MA). The cloned PCR fragments were sequenced by LGC Genomics (Berlin, Germany) using pJET1.2 forward and reverse primers, and the obtained sequence was deposited in GenBank (MK992773). A BLASTn search revealed that the sequence obtained was 100% identical to 16S rDNA of aster yellows (AY) phytoplasma (AF222063) and shared 99.95% sequence similarity with the ‘Candidatus Phytoplasma asteris’ reference strain (AP006628). A phylogenetic analysis using MEGA7 (Tamura et al. 2013) confirmed that the phytoplasma clustered with 16SrI-B strains. In subsequent experiments, three representative samples were selected for PCR amplifications of the partial elongation factor Tu (tuf), ribosomal protein (rp) operon (rps19, rpl22, and rps3), and molecular chaperonin groEL genes with primer pairs fTufl/rTufl (Schneider et al. 1997), rpF1/rpR1 (Lim and Sears 1992), and AYgroelF/AYgroelR (Mitrovic et al. 2011), respectively. The generated amplicons were cloned and sequenced (GenBank accession nos. MN526022 to MN526024), and BLAST searches of the GenBank database showed 100% identity of the tuf sequence with AY phytoplasma strain IRap (AJ271316). The rp and groEL sequences were 100% identical to six and 11 GenBank entries, respectively (e.g., Onion proliferation phytoplasma strain OnP2 for rp [GU228514] and AY phytoplasma strain AVUT for groEL [AB599686]). Comparison with the reference strains (Lee et al. 2004; Mitrovic et al. 2011) allowed classification of the phytoplasma into AY subgroups tufI-B, rpI-B, and groELI-III. To our knowledge, this is the first report of an AY phytoplasma infecting L. flos-cuculi. Identification of subgroup 16SrI-B phytoplasma infection in L. flos-cuculi pointed to its broad host range, as has been reported earlier by several investigators (e.g., Marcone et al. 2016). Considering the serious symptoms and the high incidence of the disease in nursery-grown plant material, the infection may result in economic losses to the producers.

Nuclear Accumulation of HSP70 in Mouse Skeletal Muscles in Response to Heat Stress, Aging, and Unloading With or Without Reloading.

The purpose of this study was to investigate the nuclear accumulation of heat shock protein 70 (HSP70), a molecular chaperonin in mouse skeletal muscle in response to aging, heat stress, and hindlimb unloading with or without reloading. Profiles of HSP70-specific nuclear transporter Hikeshi in skeletal muscles were also evaluated. Heat stress-associated nuclear accumulation of HSP70 was observed in slow soleus (SOL) and fast plantaris (PLA) muscles of young (10-week-old) mice. Mean nuclear expression level of HSP70 in slow medial gastrocnemius (MGAS) and PLA muscles of aged (100-week-old) mice increased ~4.8 and ~1.7 times, compared to that of young (10-week-old) mice. Reloading following 2-week hindlimb unloading caused accumulation of HSP70 in myonuclei in MGAS and PLA of young mice ( p < 0.05). However, reloading-associated nuclear accumulation of HSP70 was not observed in both types of muscles of aged mice. On the other hand, 2-week hindlimb unloading had no impact on the nuclear accumulation of HSP70 in both muscles of young and aged mice. Nuclear expression level of Hikeshi in both MGAS and PLA in mice was suppressed by aging. No significant changes in the nuclear Hikeshi in both muscles were induced by unloading with or without reloading. Results of this study indicate that the nuclear accumulation of HSP70 might show a protective response against cellular stresses in skeletal muscle and that the protective response may be suppressed by aging. Protective response to aging might depend on muscle fiber types.

Function of a thermophilic archaeal chaperonin is enhanced by electrostatic interactions with its targets

Molecular chaperonin CpkB from Thermococcus kodakarensis possesses a unique negatively charged carboxy-terminal region that functions in target protein recognition. In the present study, green fluorescent protein (GFP), 4-oxalocrotonate tautomerase (4OTA) and glutamine:fructose-6-phosphate amidotransferase (GFAT) were fused with a positively charged tag, selected using docking simulation in silico, to enhance their electrostatic interactions with CpkB. Target proteins were heated at 75°C in the presence or absence of CpkB, and the remaining enzymatic activity was measured. The half-life (t1/2) of the positively charged tagged targets was significantly longer than that of their tagless counterparts. Escherichia coli cell extracts containing heterologously expressed targets (GFP, 4OTA and GFAT and their tagged variants) were incubated at 75°C in the presence or absence of CpkB, and the proportion remaining in the soluble fraction was evaluated by SDS-PAGE. Only positively charged tagged targets remained predominantly in the soluble fraction in the presence of CpkB but not in the absence of CpkB. When tagless or negatively charged tagged targets were employed, the targets were barely detected in the soluble fraction, suggesting that CpkB protected positively charged tagged proteins more efficiently than tagless targets. Attachment of a positively charged tag may be a generally applicable method for enhancing target recognition by chaperonins carrying negatively charged carboxy-terminal regions, such as the archaeal chaperonin CpkB.

Lethal Consequences of Overcoming Metabolic Restrictions Imposed on a Cooperative Bacterial Population.

ABSTRACTQuorum sensing (QS) controls cooperative activities in many Proteobacteria. In some species, QS-dependent specific metabolism contributes to the stability of the cooperation. However, the mechanism by which QS and metabolic networks have coevolved to support stable public good cooperation and maintenance of the cooperative group remains unknown. Here we explored the underlying mechanisms of QS-controlled central metabolism in the evolutionary aspects of cooperation. In Burkholderia glumae, the QS-dependent glyoxylate cycle plays an important role in cooperativity. A bifunctional QS-dependent transcriptional regulator, QsmR, rewired central metabolism to utilize the glyoxylate cycle rather than the tricarboxylic acid cycle. Defects in the glyoxylate cycle caused metabolic imbalance and triggered high expression of the stress-responsive chaperonin GroEL. High-level expression of GroEL in glyoxylate cycle mutants interfered with the biosynthesis of a public resource, oxalate, by physically interrupting the oxalate biosynthetic enzyme ObcA. Under such destabilized cooperativity conditions, spontaneous mutations in the qsmR gene in glyoxylate cycle mutants occurred to relieve metabolic stresses, but these mutants lost QsmR-mediated pleiotropy. Overcoming the metabolic restrictions imposed on the population of cooperators among glyoxylate cycle mutants resulted in the occurrence and selection of spontaneous qsmR mutants despite the loss of other important functions. These results provide insight into how QS bacteria have evolved to maintain stable cooperation via QS-mediated metabolic coordination.

The Role of Molecular Chaperonin CCT and its Co‐chaperone PhLP1 in the Assembly of mTOR Complexes

The mechanistic target of rapamycin (mTOR) kinase signaling pathway controls cell proliferation, metabolism and survival. Mutations in components of the mTOR pathway lead to tumerogenesis, angiogenesis and metastasis and therefore are implicated in many types of cancers. mTOR signaling is mediated through two signaling complexes, namely mTORC1 (consisting of mTOR, mLST8, Raptor, Deptor and PRAS40) and mTORC2 (consisting of mTOR, mLST8, Rictor, mSin1, Deptor and Protor1). In these complexes, mTOR is the central kinase whereas the other components are accessory proteins that assist in regulation and recognition of mTOR substrates. The mechanism by which the mTOR components are assembled into functional complexes from nascent polypeptides is a fundamental question yet to be answered. We have evidence indicating that the chaperonin CCT (also called TRiC) and its co‐chaperone PhLP1 assist in mTORC1 assembly by folding two of the mTOR components, mLST8 and Raptor. CCT and PhLP1 form a ternary complex with mLST8, while Raptor binds CCT in the absence of PhLP1. Furthermore, cellular depletion of CCT or PhLP1 disrupts mTORC1 assembly, and CCT depletion inhibits mTORC2‐mediated phosphorylation of Ser473 of AKT. In contrast, the mTORC2 subunit Rictor, which is structurally homologous to Raptor, does not bind CCT. These results suggest that CCT and PhLP1 contribute to mLST8 folding and assembly into mTORC1 and mTORC2, while CCT alone assists in Raptor folding and assembly into mTORC1. Thus, the CCT chaperonin and its co‐chaperones may represent new physiological or therapeutic targets to regulate mTOR signaling.Support or Funding InformationNIH Grants: EY 012287, GM 078550, Brigham Young University Cancer Research Center

Enhanced expression of soluble human papillomavirus L1 through coexpression of molecular chaperonin in Escherichia coli

The major recombinant capsid protein L1 of human papillomavirus (HPV) is widely used to produce HPV prophylactic vaccines. However, the quality of soluble and active expression of L1 in Escherichia coli was below the required amount. Coexpression with the chaperonin GroEL/ES enhanced L1 expression. Overexpressing GroEL/ES increased the soluble expression level of glutathione S-transferase-fused L1 (GST-L1) by approximately ∼3 fold. The yield of HPV type 16 L1 pentamer (L1-p) was ∼2 fold higher than that in a single expression system after purification through size-exclusion chromatograph. The expression and purification conditions were then optimized. The yield of L1-p was enhanced by ∼5 fold, and those of HPV types 18 and 58 L1-p increased by ∼3 and ∼2 folds, respectively, compared with that in the single expression system. Coexpressing the mono-site mutant HPV16 L1 L469A with GroEL/ES increased L1-p yield by ∼7 fold compared with strains expressing the wild-type L1 gene. L1-p was then characterized using circular dichroism spectra, UV–vis cloud point, dynamic light scattering and transmission electron microscope analyses. Results indicated that the conformation and biological characteristics of L1-p were identical to that of native L1. Hence, overexpressing chaperonin in E. coli can increase the expression level of GST-L1 and L1-p production after purification. This finding may contribute to the development of a platform for prophylactic HPV vaccines.

Multigene Sequence Analysis of Aster Yellows Phytoplasma Associated with Primrose Yellows

Primula acaulis (L.) Hill. plants showing stunting, leaf-yellowing and virescence were first discovered in the Czech Republic. Polymerase chain reactions with subsequent restriction fragment length polymorphism analyses and sequencing enabled classification of the detected phytoplasmas into the aster yellows group, ribosomal subgroup 16SrI-B, tufI-B, rpI-B, groELIB-III and SecY-IB subgroups. Phylogeny of the 16S rRNA gene sequences as well as sequence analysis of several chromosomal regions, such as the 16S-23S ribosomal operon, ribosomal proteins, spc ribosomal protein operon, genes for elongation factor EF-Tu, molecular chaperonin large subunit GroEL, immunodominant membrane protein, ribosome recycling factor, urydilate kinase, ATP- and Zn2+-dependent proteases not only confirmed its affiliation with the ‘Candidatus Phytoplasma asteris’ species but also enabled its detailed molecular characterization. The less researched regions of phytoplasma genome (amp, adk, hflB, pyrH-frr genes) could be valuable as additional markers for phytoplasma through differentiation especially within the 16SrI-B ribosomal subgroup.

Fitness Trade-Offs Determine the Role of the Molecular Chaperonin GroEL in Buffering Mutations.

Molecular chaperones fold many proteins and their mutated versions in a cell and can sometimes buffer the phenotypic effect of mutations that affect protein folding. Unanswered questions about this buffering include the nature of its mechanism, its influence on the genetic variation of a population, the fitness trade-offs constraining this mechanism, and its role in expediting evolution. Answering these questions is fundamental to understand the contribution of buffering to increase genetic variation and ecological diversification. Here, we performed experimental evolution, genome resequencing, and computational analyses to determine the trade-offs and evolutionary trajectories of Escherichia coli expressing high levels of the essential chaperonin GroEL. GroEL is abundantly present in bacteria, particularly in bacteria with large loads of deleterious mutations, suggesting its role in mutational buffering. We show that groEL overexpression is costly to large populations evolving in the laboratory, leading to groE expression decline within 66 generations. In contrast, populations evolving under the strong genetic drift characteristic of endosymbiotic bacteria avoid extinction or can be rescued in the presence of abundant GroEL. Genomes resequenced from cells evolved under strong genetic drift exhibited significantly higher tolerance to deleterious mutations at high GroEL levels than at native levels, revealing that GroEL is buffering mutations in these cells. GroEL buffered mutations in a highly diverse set of proteins that interact with the environment, including substrate and ion membrane transporters, hinting at its role in ecological diversification. Our results reveal the fitness trade-offs of mutational buffering and how genetic variation is maintained in populations.

Skeletal muscle heat shock protein 60 increases after endurance training in mice and induces peroxisome proliferation-activated receptor-γ coactivator-1 α1 expression

Heat shock protein (Hsp60) is a mitochondrial chaperonin whose unconventional cellular localizations and functions are discovered day by day. In the present study, the levels of Hsp60 in fibres of the soleus muscle and its correlation to the expression of four isoforms of peroxisome proliferation-activated receptor-γ (PPAR-γ) coactivator-1α (PGC1α) were investigated in 72 young (7-weeks old) healthy male mice (BALB/c AnNHsd) at baseline and after completing a 6-week endurance training program. The mice were assigned to one of the two experimental groups: SED (sedentary) or TR (trained). Short-term overexpression of hsp60, achieved by in vitro plasmid transfection, was then performed to determine whether this chaperonin could have a role in the activation of the expression levels of PGC-1α isoforms. The levels of Hsp60 protein were fibre-type specific in the posterior muscles at baseline, and endurance training increased its content in type I muscle fibers. Concomitantly with the increased levels of Hsp60 released in the blood stream of trained mice, mitochondrial copy number and the expression of three isoforms of PGC-1α increased. Overexpressing hsp60 in cultured myoblasts induced only the expression of PGC-1 α1, letting us suppose a direct correlation between Hsp60 overexpression and PGC-1 α1 activation. Overall, these results suggest that during endurance training Hsp60 is upregulated and activates the mitochondrial biogenesis pathway, probably as a response to the oxidative stress induced by exercise. This study reveals a molecular response of skeletal muscle to a mechanical stress induced by training which involves the molecular chaperonin Hsp60 and the transcriptional co-activator PGC-1 α1. The role of these proteins in aerobic adaptation and pathological conditions as cancer cachexia warrants further investigations.

Mechanistic Insights into the Folding of Knotted Proteins In Vitro and In Vivo

The importance of knots and entanglements in biological systems is increasingly being realized and the number of proteins with topologically complex knotted structures has risen. However, the mechanism as to how these proteins knot and fold efficiently remains unclear. Using a cell-free expression system and pulse-proteolysis experiments, we have investigated the mechanism of knotting and folding for two bacterial trefoil-knotted methyltransferases. This study provides the first experimental evidence for a knotting mechanism. Results on fusions of stable protein domains to N-terminus, C-terminus or both termini of the knotted proteins clearly demonstrate that threading of the nascent chain through a knotting loop occurs via the C-terminus. Our results strongly suggest that this mechanism occurs even when the C-terminus is severely hindered by the addition of a large stable structure, in contrast to some simulations indicating that even the folding pathways of knotted proteins have some plasticity. The same strategy was employed to probe the effects of GroEL-GroES. In this case, results suggest active mechanisms for the molecular chaperonin. We demonstrate that a simple model in which GroEL-GroES sterically confines the unknotted polypeptide chain thereby promoting knotting is unlikely, and we propose two alternatives: (a) the chaperonin facilitates unfolding of kinetically and topologically trapped intermediates or (b) the chaperonin stabilizes interactions that promote knotting. These findings provide mechanistic insights into the folding of knotted proteins both in vitro and in vivo, thus elucidating how they have withstood evolutionary pressures regardless of their complex topologies and intrinsically slow folding rates. [Display omitted]

Polyglutamine Flanking Regions in Huntingtin Highlight Key Structural Intermediates Relevant for Molecular Chaperone Interaction and Huntington's Disease Pathogenesis

Huntington's Disease (HD) is a neurodegenerative disorder caused by an expansion in the polyglutamine (polyQ) tract of the Huntingtin (Htt) protein. The clinical hallmark of this mutation is the accumulation of amyloid Htt aggregates in neurons. While Htt aggregation is highly correlated with the length of this polyQ tract both in vitro and in vivo, recent studies suggest that the regions flanking the polyQ tract can influence Htt aggregation and toxicity independent of polyQ length. In addition, it is well established that two polyQ flanking regions in exon1 of Htt, the N-terminal first 17 amino acids (N17) and a proline-rich region (PolyPro), influence Htt aggregation propensity. Here, we show that the N17 and PolyPro regions of Htt dramatically influence the rate of the Htt aggregation pathway. We also show that mutations within these polyQ-flanking regions alter Htt toxicity in a brain slice model. The influence of these flanking regions on the heterogeneous distribution of the aggregate species may account for these differences in toxicity. This theory is increasingly relevant in light of recent thought that Htt toxicity may derive from a species other than the amyloid fibril itself. Finally, previous work has shown that the N17 region of Htt interacts with the molecular chaperonin TRiC, and this interaction exerts a protective effect against both Htt aggregation and toxicity. An understanding how these flanking regions influence Htt aggregation will inform how TRiC works through the N17 element. Together, this information can provide the basis to design HD therapeutics that exploit these flanking regions to suppress HD pathogenesis.

Biphenyl- and carvone-induced protein expression patterns in Rhodococcus sp. ACS

Protein expression patterns in the polychlorinated biphenyl (PCB)-degrading Rhodococcussp. strain ACS were examined following growth on two substrates capable of inducing the enantioselective biotransformation of PCBs via different degradation pathways. Eleven inducible proteins were identified by SDS-PAGE and characterized by LC-MS/MS. Four of the peptides, a spore coat protein, an extracellular serine protease, a spoVP, and a molecular chaperonin from Bacillus subtilis, were identified as being unique to biphenyl-induced cells, whereas anextracellular serine protease from B. subtilis was identified as being unique to carvone-induced cells. None of the peptides identified had sequences that corresponded to known dioxygenases or other PCB-degrading enzymes of this Gram- positive bacterium, suggesting that the identified induced proteins may be involved in either PCB degradation or adaptive responses that protect cells from toxicity.

FSUB: Normal Mode Analysis with Flexible Substructures

In this paper, we report a novel normal-mode analysis for supramolecular complexes, named fSUB. The method models a complex as a group of flexible substructures. The low-frequency substructure modes are first determined with substructures in isolation, and the motions of the whole complex are then calculated on the basis of substructure modes and substructure-substructure interactions. The calculation of modes in fSUB requires modal analysis without initial energy minimization, which is essential for maintaining energetic and structural consistency between substructures and whole complex. Compared with other coarse-grained methods, such as the RTB method, fSUB delivers much more accurate modes for the complex and allows for the choice of much larger substructures. The method can also accommodate any type of substructure arrangement including covalent bonds across the interface. In tests on molecular chaperonin GroEL (7350 residues) and HK97 capsid complex (118,092 residues), fSUB was shown to be much more efficient in terms of combined accuracy and demand of computing resources. Our results clearly demonstrated the vital importance of including substructure flexibility in complex modal analysis, as the deformational patterns of substructures were found to play an important role even in the lowest frequency modes of the whole complex.

Detection and Differentiation of Non-Emetic and Emetic Bacillus cereus Strains in Food by Real-Time PCR

Duplex real-time polymerase chain reaction (PCR) method was developed for direct detection and identification of non-emetic and emetic Bacillus cereus strains in foods without enrichment. Primers and TaqMan probes were designed for molecular chaperonin gene groEL and cereulide synthetase gene ces. A total of 62 B. cereus strains, of which 59 were non-emetic and 3 emetic, were found positive by groEL-specific conventional PCR. Three emetic strains were found by ces-specific PCR to be positive, whereas 59 non-emetic strains were negative. Ten strains other than B. cereus were all negative by both groEL-specific and ces-specific PCR assays. The limits of detection of the duplex PCR assays for both non-emetic and emetic strains from their pure cultures were 3× 10° CFU/reaction. A total of 10 doenjang (traditional Korean fermented soybean paste) samples were analyzed simultaneously by real-time PCR assay and analytical profile index (API) test kits. All tested samples were positive for B. cereus contamination except two samples. The result of the real-time PCR assay was consistent with that of the API test. Among the eight positive samples, six and two samples were contaminated with non-emetic and emetic strains, respectively. The results suggest that the real-time PCR method developed in the present study may be useful for the direct detection and differentiation of B. cereus strains in foods.

Heterologous production of active ribonuclease inhibitor in Escherichia coli by redox state control and chaperonin coexpression

BackgroundEukaryotic Ribonuclease inhibitor (RI), belonging to the RNH1 family, is distinguished by unique features - a high sensitivity to oxidation due to the large number of reduced cysteins and a high hydrophobicity, which made most production approaches so far unsuccessful or resulted in very low yields. In this work efficient in vivo folding of native RI in the Escherichia coli cytoplasm was obtained by external addition of a reducing agent in tandem with oxygen limitation and overproduction of a molecular chaperonin. After optimisation of the production conditions in the shake flask scale the process was scaled up to high cell densities by applying a glucose limited fed-batch procedure.ResultsRI production in a T7 RNA polymerase based system results in accumulation of aggregated inactive product in inclusion bodies. Combination of addition of the reductant DTT, low production temperature and coexpression of the chaperonin GroELS resulted in high level production of approximately 25 mg g-1 CDW active RI in E. coli ER2566 pET21b, corresponding to approximately 800 kU g-1 cell wet weight. Further conditional screening under fed-batch-like conditions with the EnBase® technology and scale up into the bioreactor scale resulted in an efficient high cell density glucose and oxygen limited fed-batch process with a final cell dry weight of 25 g L-1 and a total RI yield of app. 625 mg L-1 (volumetric activity of 80,000 kU L-1). The E. coli based production constructs showed a very high robustness. The recombinant culture maintained its productivity despite the combination of the toxic growth conditions, the substrate limited production mode in tandem with a high level expression of several recombinant proteins, the set of molecular chaperonins and the target protein (RI).ConclusionsHigh level production of active RI in E. coli in a T7 RNA polymerase expression system depends on the following factors: (i) addition of a reducing agent, (ii) low production temperature, (iii) oxygen limitation, and (iii) co-overexpression of the chaperonin GroELS. The study indicates the strength of applying fed-batch cultivation techniques for the efficient optimisation of production factors already at the screening stage for fast and straight forward bioprocess development even for target proteins which show a complex folding behaviour. In our case none of the approaches alone would have resulted in significant accumulation of active RI.

Chaperonin 10 as a putative modulator of multiple Toll-like receptors for the treatment of inflammatory diseases

Chaperonin 10 (Cpn10) is a mitochondrial protein that associates with chaperonin 60 (Cpn60) to form a multimeric molecular chaperonin complex whose main function is to promote the correct native folding of newly synthesized polypeptides within mitochondria. Cpn10 is also released extracellularly and can be found as an early pregnancy factor with immunomodulatory properties in the serum and urine of pregnant women. Furthermore, studies in animal models along with a recent clinical trial in rheumatoid arthritis patients have suggested that exogenously administered Cpn10 may hold potential for the treatment of inflammatory and autoimmune diseases. Investigations of the mechanism of immunosuppressive action of Cpn10 that focused on a possible functional modulation of the Toll-like receptor (TLR) system, known to play a central role in innate immunity and inflammation, have shown that Cpn10 can inhibit pro-inflammatory cytokine production mediated via TLR4 in macrophages. In these three patent applications by CBIO Ltd, the ability of Cpn10 to modulate TLR function is substantiated and extended to TLR2, TLR3, TLR6, TLR7 and TLR9. In addition, Cpn10-derived polypeptides that possess immunomodulatory activity, but little or no protein folding activity in the presence of Cpn60, are identified. Thirdly, it is shown that Cpn10 can ameliorate airway inflammation in a sheep model of asthma. Overall, these findings provide additional evidence that Cpn10 may participate in a novel pathway of immunoregulation. However, the exact molecular mechanisms involved in this pathway remain to be characterized. The efficacy and safety of Cpn10 as a therapeutic immunomodulator also need to be further defined.

Testing the Neutral Fixation of Hetero-Oligomerism in the Archaeal Chaperonin CCT

The evolutionary transition from homo-oligomerism to hetero-oligomerism in multimeric proteins and its contribution to function innovation and organism complexity remain to be investigated. Here, we undertake the challenge of contributing to this theoretical ground by investigating the hetero-oligomerism in the molecular chaperonin cytosolic chaperonin containing tailless complex polypeptide 1 (CCT) from archaea. CCT is amenable to this study because, in contrast to eukaryotic CCTs where sub-functionalization after gene duplication has been taken to completion, archaeal CCTs present no evidence for subunit functional specialization. Our analyses yield additional information to previous reports on archaeal CCT paralogy by identifying new duplication events. Analyses of selective constraints show that amino acid sites from 1 subunit have fixed slightly deleterious mutations at inter-subunit interfaces after gene duplication. These mutations have been followed by compensatory mutations in nearby regions of the same subunit and in the interface contact regions of its paralogous subunit. The strong selective constraints in these regions after speciation support the evolutionary entrapment of CCTs as hetero-oligomers. In addition, our results unveil different evolutionary dynamics depending on the degree of CCT hetero-oligomerism. Archaeal CCT protein complexes comprising 3 distinct classes of subunits present 2 evolutionary processes. First, slightly deleterious and compensatory mutations were fixed neutrally at inter-subunit regions. Second, sub-functionalization may have occurred at substrate-binding and adenosine triphosphate-binding regions after the 2nd gene duplication event took place. CCTs with 2 distinct types of subunits did not present evidence of sub-functionalization. Our results provide the 1st in silico evidence for the neutral fixation of hetero-oligomerism in archaeal CCTs and provide information on the evolution of hetero-oligomerism toward sub-functionalization in archaeal CCTs.

Properties of the α subunit of a Chaperonin from the hyperthermophilic CrenarchaeonAeropyrum pernixK1

The gene encoding for a putative thermosome from the hyperthermophilic crenarchaeon Aeropyrum pernix K1 (ApcpnA) was cloned and the biochemical characteristics of the resulting recombinant protein were examined. The gene (accession no. APE0907) from A. pernix K1 showed some homology with other group II chaperonins from archaea. The recombinant ApcpnA protein has a molecular mass of 60 kDa, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and exhibited ATPase activity with an optimum temperature and pH of 90 degrees C and 5.0, respectively. The ATPase activity was found to be dependent on manganese and potassium ions, but not magnesium ion. The K(m) for ATP at pH 5.0 and 90 degrees C was 10.04 (+/- 1.31) microM, and k(cat) was determined to be 2.21 (+/- 0.11) min(-1) for the ApcpnA monomer. The recombinant ApcpnA prevents thermal aggregation of bovine rhodanese and enhances the thermal stability of alcohol dehydrogenase in vitro, indicating that the protein is suitable as a molecular chaperonin in the high-temperature environment.