Functional fusion mutant of Candida antarctica lipase B (CalB) expressed in Escherichia coli.

Candida antarctica lipase B (CalB) was functionally expressed in the cytoplasm of Escherichia coli Origami(DE3) with the N-terminus fusion of E. coli endogenous proteins. The previously-identified stress responsive proteins through comparative proteome analyses such as malate dehydrogenase (Mdh), spermidine/putrescine-binding periplasmic protein (PotD), and FKBP-type peptidyl-prolyl cis-trans isomerase (PPIases) (SlyD) dramatically increased the solubility of CalB in E. coli cytoplasm when used as N-terminus fusion partners. We demonstrated that Mdh, PotD, and SlyD were powerful solubility enhancers that presumably facilitated the protein folding of CalB. Moreover, among the various fusion mutants, Mdh-CalB showed the highest hydrolytic activity and was as biologically active as standard CalB. Similarly to the previous report, the electrophoretic properties of CalB indicate that CalB seems to form dimer-based oligomer structures. We evaluated the structural compatibility between the fusion partner protein and CalB, which seems to be of crucial importance upon the bioactive dimer formation of CalB and might affect the substrate accessibility to the enzyme active site, thereby determining the biological activities of the fusion mutants.

Transport proteins PotD and Crr of Escherichia coli, novel fusion partners for heterologous protein expression.

The Escherichia coli proteome response to the stressor GdnHCl was analyzed through 2-dimensional gel electrophoresis (2-DE). We identified PotD (spermidine/putrescine-binding periplasmic protein) and Crr [glucose-specific phosphotransferase (PTS) enzyme IIA component] as a stress-responsive protein. Even under a stress situation where the total number of soluble proteins decreased by about 10%, 3.5- and 2.2-fold increase was observed in the synthesis of PotD and Crr, respectively. As fusion partners, PotD and Crr dramatically increased the solubility of many aggregation-prone heterologous proteins [e.g. human minipro-insulin (mp-INS), human epidermal growth factor (EGF), human prepro-ghrelin (ppGRN), human interleukin-2(hIL-2), human activation induced cytidine deaminase (AID), human glutamate decarboxylase (GAD(448-585)), Pseudomonas putida cutinase (CUT), human ferritin light chain (hFTN-L), human granulocyte colony-stimulating factor (G-CSF), and cold autoinflammatory syndrome1 protein (NALP3) Nacht domain (NACHT)] in the E. coli cytoplasm. Presumably PotD and Crr were very effective in shielding interactive surfaces of heterologous proteins associated with non-specific protein-protein interactions leading to the formation of inclusion bodies most likely due to intrinsic high folding efficiency, chaperone-like activity, or a combination of both factors. Both the stress-induced proteins were well suited for the production of a biologically active fusion mutant of P. putida cutinase that can be expected to be of biotechnological and commercial interest.

Solubility enhancement of aggregation-prone heterologous proteins by fusion expression using stress-responsive Escherichia coli protein, RpoS.

BACKGROUND: The most efficient method for enhancing solubility of recombinant proteins appears to use the fusion expression partners. Although commercial fusion partners including maltose binding protein and glutathione-S-transferase have shown good performance in enhancing the solubility, they cannot be used for the proprietory production of commercially value-added proteins and likely cannot serve as universal helpers to solve all protein solubility and folding issues. Thus, novel fusion partners will continue to be developed through systematic investigations including proteome mining presented in this study. RESULTS: We analyzed the Escherichia coli proteome response to the exogenous stress of guanidine hydrochloride using 2-dimensional gel electrophoresis and found that RpoS (RNA polymerase sigma factor) was significantly stress responsive. While under the stress condition the total number of soluble proteins decreased by about 7 %, but a 6-fold increase in the level of RpoS was observed, indicating that RpoS is a stress-induced protein. As an N-terminus fusion expression partner, RpoS increased significantly the solubility of many aggregation-prone heterologous proteins in E. coli cytoplasm, indicating that RpoS is a very effective solubility enhancer for the synthesis of many recombinant proteins. RpoS was also well suited for the production of a biologically active fusion mutant of Pseudomonas putida cutinase. CONCLUSION: RpoS is highly effective as a strong solubility enhancer for aggregation-prone heterologous proteins when it is used as a fusion expression partner in an E. coli expression system. The results of these findings may, therefore, be useful in the production of other biologically active industrial enzymes, as successfully demonstrated by cutinase.

A novel approach to ultrasensitive diagnosis using supramolecular protein nanoparticles.

We report on the ultrasensitive protein nanoprobe system that specifically captures disease marker (autoantibodies of Type I diabetes in this case) with attomolar sensitivity. The system relies on supramolecular protein nanoparticles that bind a specific antibody [65 kDa glutamate decarboxylase (GAD65)-specific autoantibody, i.e., the early marker of Type I diabetes]. The ultrasensitive detection of early marker of Type I diabetes during the early phase of pancreatic beta-cell destruction is important because individuals at high risk of developing Type I diabetes can be identified several years before the clinical onset of the ailment. The bacterial expression of chimera genes encoding N-[human ferritin heavy chain (hFTN-H)]::[specific antigenic epitope]-C produces supramolecular nanoparticles with uniform diameters (10-15 nm), owing to self-assembly activity of hFTN-H. Each nanoparticle, formed by intermolecular self-assembly between the chimera protein molecules, is subjected to carrying a large number (presumably, 24) of epitopes with a homogeneous and stable conformation per autoantibody binding, thereby allowing substantial enhancement of sensitivity. The sensitivity was finally boosted to 3 attomolar concentration of the autoantibodies, 4-9 orders of magnitude more sensitive than conventional immunoassays. Also, this ultrasensitive protein nanoprobe successfully detected natural autoantibodies in the sera from Type I diabetic patients. The attomolar sensitivity was successfully reproduced on the detection of other antibodies, i.e., monoclonal antibodies against hepatitis B surface antigen. With the two antibody markers above, the feasibility of simultaneous and multiplexing-mode detection was also demonstrated.

Solubilization of aggregation-prone heterologous proteins by covalent fusion of stress-responsive Escherichia coli protein, SlyD.

The proteome profile of Escherichia coli BL21(DE3) generated in response to heat shock stress was analyzed by two-dimensional electrophoresis (2-DE), wherein we identified a FKBP-type peptidyl-prolyl cis-trans isomerse (PPIases), SlyD, as a stress-responsive (i.e. aggregation-resistant) protein. Even under an imposed severe stress condition where 29 out of 858 soluble proteins were totally eliminated and the synthesis levels of 171 proteins decreased over 5-fold, a 3.37-fold increase induced by heat shock treatment was observed in the synthesis level of SlyD compared with a non-stress condition. As a fusion partner, as well as solubility enhancer, SlyD facilitated folding and significantly increased the solubility of many aggregation-prone heterologous proteins in E. coli cytoplasm. SlyD was very effective in sequestering interactive surfaces of heterologous proteins associated with non-specific protein-protein interactions and the formation of inclusion bodies, most likely as a result of intrinsic folding efficiencies and/or chaperone-like activities. SlyD was also shown to be suitable for the production of a biologically active fusion mutant of Pseudomonas putida cutinase that is of considerable biotechnological and commercial interest.

Enhanced solubility of heterologous proteins by fusion expression using stress-induced Escherichia coli protein, Tsf.

Through two-dimensional electrophoresis, Escherichia coli proteome response to a protein denaturant, guanidine hydrochloride, was analyzed and elongation factor Ts (Tsf) detected as a stress-induced protein. Many host proteins aggregated, or their synthesis levels decreased significantly under conditions of protein denaturation as 34 out of 699 soluble proteins knocked out and 63 proteins decreased by over 2.5-fold. Interestingly, the expression level of Tsf increased 1.61-fold compared with a nonstress condition. Contrary to direct expression, various heterologous proteins were solubly expressed in E. coli when subjected to N-terminus fusions of Tsf. Owing most likely to an intrinsic high folding efficiency, Tsf seemed to play critical roles in sequestering interactive surfaces of heterologous proteins from nonspecific protein-protein interactions leading to formation of inclusion bodies. It has been also demonstrated that Tsf is effective in aiding the production of a biologically active bacterial cutinase, which could be of interest to biotechnology and commercial applications.

Multiplex diagnosis of viral infectious diseases (AIDS, hepatitis C, and hepatitis A) based on point of care lateral flow assay using engineered proteinticles.

Lateral flow assay (LFA) is an attractive method for rapid, simple, and cost-effective point of care diagnosis. For LFA-based multiplex diagnosis of three viral intractable diseases (acquired immune deficiency syndrome and hepatitis C and A), here we developed proteinticle-based 7 different 3D probes that display different viral antigens on their surface, which were synthesized in Escherichia coli by self-assembly of human ferritin heavy chain that was already engineered by genetically linking viral antigens to its C-terminus. Each of the three test lines on LFA strip contains the proteinticle probes to detect disease-specific anti-viral antibodies. Compared to peptide probes, the proteinticle probes were evidently more sensitive, and the proteinticle probe-based LFA successfully diagnosed all the 20 patient sera per each disease without a false negative signal, whereas the diagnostic sensitivities in the peptide probe-based LFAs were 65-90%. Duplex and triplex assays performed with randomly mixed patient sera gave only true positive signals for all the 20 serum mixtures without any false positive signals, indicating 100% sensitivity and 100% specificity. It seems that on the proteinticle surface the antigenic peptides have homogeneous orientation and conformation without inter-peptide clustering and hence lead to the enhanced diagnostic performance with solving the problems of traditional diagnostic probes. Although the multiplex diagnosis of three viral diseases above was demonstrated as proof-of-concept here, the proposed LFA system can be applied to multiplex point of care diagnosis of other intractable diseases.

Proteinticle engineering for accurate 3D diagnosis.

In nature certain proteins are self-assembled inside cells to form nanoscale particles (named "proteinticles") with constant structure and surface topology. Unlike chemically synthesized nanomaterials (e.g., various metal, carbon, and polymer nanoparticles), a variety of functional proteinticles can be easily created through genetic modification of the proteinticle surface, i.e., by adding or inserting specified proteins/peptides to the N- or C-terminus or the internal region of the protein constituent. Here we present proteins/peptides that recognize disease-specific antibodies on the surface of human ferritin based proteinticles for accurate 3D diagnosis of human autoimmune and infectious diseases. The surface display of the extracellular domain of myelin oligodendrocyte glycoprotein (MOG) with native conformation successfully discriminated between autoantibodies to native or denatured MOG, leading to the reliable diagnosis of multiple sclerosis with enhanced accuracy. Also we simultaneously displayed different antigenic peptides from hepatitis C virus (HCV) on the same proteinticle surface with modulating the composition of each peptide. The proteinticles with the heterogeneous peptide surface detected anti-HCV antibodies in patient sera with 100% accuracy. The proposed method of proteinticle engineering can be applied in general to the sensitive and specific diagnosis of many other human diseases.

Human G-CSF synthesis using stress-responsive bacterial proteins.

We previously reported that under the stress condition caused by the addition of 2-hydroxyethyl disulfide, a thiol-specific oxidant, to growing cultures of Escherichia coli BL21(DE3), a population of stress-responsive proteins [peptidyl-prolyl cis-trans isomerase B (PpiB), bacterioferritin (Bfr), putative HTH-type transcriptional regulator yjdC (YjdC), dihydrofolate reductase (FolA), chemotaxis protein cheZ (CheZ), and glutathione synthetase (GshB)] were significantly upregulated when compared with the nonstress condition. When those stress-responsive proteins were used as fusion partners for the expression of human granulocyte colony-stimulating factor (hG-CSF), the solubility of hG-CSF was dramatically enhanced in E. coli cytoplasm, whereas almost all of the directly expressed hG-CSF were aggregated to inclusion bodies. In addition, the spectra of circular dichroism measured with the purified hG-CSF were identical to that of standard hG-CSF, implying that the synthesized hG-CSF has native conformation. These results indicate that the bacterial stress-responsive proteins could be potent fusion expression partners for aggregation-prone heterologous proteins in E. coli cytoplasm.

Multiple stressor-induced proteome responses of Escherichia coli BL21(DE3).

Through 2-DE based quantitative proteomics, the dynamic characteristics of overall proteome profiles of Escherichia coli BL21(DE3) were systematically analyzed in the presence of four different stressors. Dithiothreitol and 2-hydroxyethyl disulfide are a reducing and an oxidizing agent, respectively, which disturb the redox balance in cytoplasm, while guanidine hydrochloride and heat shock are protein denaturants influencing protein folding. Heat shock proteins/foldases, transcription/translation-related proteins, various metabolic enzymes, and other stress regulatory proteins were found to be significantly up-regulated in response to the stressors. Heat shock proteins and translation-related proteins were generally responsive to almost all stress conditions. Two stressors, oxidative stress and guanidine hydrochloride-derived protein denaturation commonly induced the up-regulation of proteins related to transcription, whereas metabolic enzymes showed stress responses especially to the treatment of guanidine hydrochloride and heat shock. Similarities and differences of stress responses and protein-protein interactions of 80 proteins were systematically compared, and of special note, proteome-based stress-responsive proteins identified in the present study included 26 proteins that are being reported for the first time. The quantitative and systematic proteome analyses that we have performed provide more detailed information on E. coli BL21(DE3), a widely used host strain for recombinant protein overexpression.

Analysis and characterization of hepatitis B vaccine particles synthesized from Hansenula polymorpha.

The biochemical and physical properties of hepatitis B virus (HBV) small surface antigen (S-HBVsAg) from Berna Biotech Korea Corp. were systematically analyzed and characterized. Through various electrophoresis and immunoblotting assay of S-HBVsAg and its proteolytic products, it was confirmed that the S-HBVsAg vaccine particles are present in the form of covalent multimers that are assembled via strong intermolecular disulfide bonds. The S-HBVsAg particles contain no N-glycosylation moiety but some O-glycosidically linked mannoses. Evidently from N-terminus sequencing of both monomers and dimers that are formed by complete and partial reduction, respectively, of the S-HBVsAg particles under reducing SDS-PAGE condition, it is evident that each polypeptide within S-HBVsAg particles has authentic sequence of N-terminus. Denaturation plot shows that the S-HBVsAg vaccine particles were extremely stable especially in the solution with high acidity. This stability property of S-HBVsAg vaccine particles could provide very useful information for the optimization of the downstream process of recombinant S-HBVsAg particles synthesized from yeast cultures.

Escherichia coli malate dehydrogenase, a novel solubility enhancer for heterologous proteins synthesized in Escherichia coli.

Using 2-dimensional gel electrophoresis, the Escherichia coli proteome response to a heat-shock stress was analyzed and a 1.6-fold increase of malate dehydrogenase was observed even under the heat-shock condition where the total number of soluble proteins decreased by about 5%. We subsequently demonstrated that, as an N-terminus fusion expression partner, malate dehydrogenase facilitated the folding of, and dramatically increased the solubility of, many aggregation-prone heterologous proteins in E. coli cytoplasm. Therefore, malate dehydrogenase is well suited for production of a biologically active fusion mutant of cutinase (Pseudomonas putida origin) that is currently of considerable to biotechnology and commercial industries.

Pseudozyma jejuensis sp. nov., a novel cutinolytic ustilaginomycetous yeast species that is able to degrade plastic waste.

An ustilaginomycetous anamorphic yeast, isolated from orange leaves on Jeju island in South Korea, represents a novel Pseudozyma species according to morphologic and physiologic findings and molecular taxonomic analysis using the D1/D2 domains of the large subunit (26S) rRNA gene and the internally transcribed spacer (ITS) 1+2 regions. The name Pseudozyma jejuensis sp. nov. is proposed for this novel species, with OL71(T) (=KCTC 17482(T)=CBS 10454(T)) as type strain. In the present study, we have also demonstrated that Pseudozyma jejuensis OL71 is capable of producing cutinase and degrading polycaprolactone. These results suggest that Pseudozyma jejuensis or its cutinase may be useful for the biological degradation of plastic waste.

Enhanced stability of heterologous proteins by supramolecular self-assembly.

Recently, we reported on the dual function of human ferritin heavy chain (hFTN-H) used for the fusion expression and solubility enhancement of various heterologous proteins: (1) high-affinity interaction with HSP70 chaperone DnaK and (2) formation of self-assembled supramolecules with limited and constant sizes. Especially the latter, the self-assembly function of hFTN-H is highly useful in avoiding the undesirable formation of insoluble macroaggregates of heterologous proteins in bacterial cytoplasm. In this study, using enhanced green fluorescent protein (eGFP) and several deletion mutants of Mycoplasma arginine deiminase (ADI(132-410)) as reporter proteins, we confirmed through TEM image analysis that the recombinant fusion proteins (hFTN-H::eGFP and hFTN-H::ADI(132-410)) formed intracellular spherical particles with nanoscale diameter ( approximately 10 nm), i.e., noncovalently cross-linked supramolecules. Surprisingly, the supramolecular eGFP and ADI showed much enhanced stability in bioactivity. That is, the activity level was much more stably maintained for the prolonged period of time even at high temperature, at high concentration of Gdn-HCl, and in wide range of pH. The stability enhancement by supramolecular self-assembly may make it possible to utilize the protein supramolecules as novel means for drug delivery, enzymatic material conversion (biotransformation), protein chip/sensor, etc. where the maintenance of protein/enzyme stability is strictly required.

The influence of N-glycosylation and C-terminal sequence on secretion of HBV large surface antigen from S. cerevisiae.

In Saccharomyces cerevisiae, we synthesized and secreted L-HBVsAg (named as pre-S(Met1 to Asn174)::S(Met175 to Ile400)) and three mutants, i.e., pre-S degree degree::S (Asn15Gln and Asn123Gln), pre-S degree degree::S degree (Asn15Gln, Asn123Gln, and Asn320Gln), and pre-S degree degree::S degree degree (Asn15Gln, Asn123Gln, Asn233Gln, and Asn320Gln). All of the secreted pre-S::S was N-glycosylated, i.e., hyper-mannosylated. In the secretion of pre-S degree degree::S and pre-S degree degree::S degree, besides the hyper-mannosylated form, another immunoreactive protein with much lower molecular mass was observed, which seems to be unglycosylated form of pre-S degree degree::S and pre-S degree degree::S degree. Only a part of the secreted pre-S degree degree::S or pre-S degree degree::S degree molecules was N-glycosylated, and the site for the partial N-glycosylation seems to be Asn233 in S-antigen region. Compared to the N-glycosylated pre-S degree degree::S and pre-S degree degree::S degree, pre-S degree degree::S degree degree (non-N-glycosylated mutant) was secreted with lower secretion efficiency but showed apparent immunoreactivity to anti-S antigen monoclonal Ab. Interestingly, unlike pre-S degree degree::S degree degree with authentic C-terminus, the recombinant pre-S degree degree::S degree degree with C-terminal myc or poly-histidine tag (pre-S degree degree::S degree degree::tag) was almost all aggregated into insoluble proteins in the intracellular region. Conclusively, the C-terminal sequence and glycosylation in S-antigen region seem to be of crucial importance in determining the secretion efficiency of L-HBVsAg in S. cerevisiae.