Jin Seung Park

A highly sensitive and selective diagnostic assay based on virus nanoparticles.

Early detection of the protein marker troponin I in patients with a higher risk of acute myocardial infarction can reduce the risk of death from heart attacks. Most troponin assays are currently based on the conventional enzyme linked immunosorbent assay and have detection limits in the nano- and picomolar range. Here, we show that by combining viral nanoparticles, which are engineered to have dual affinity for troponin antibodies and nickel, with three-dimensional nanostructures including nickel nanohairs, we can detect troponin levels in human serum samples that are six to seven orders of magnitude lower than those detectable using conventional enzyme linked immunosorbent assays. The viral nanoparticle helps to orient the antibodies for maximum capture of the troponin markers. High densities of antibodies on the surfaces of the nanoparticles and nanohairs lead to greater binding of the troponin markers, which significantly enhances detection sensitivities. The nickel nanohairs are re-useable and can reproducibly differentiate healthy serum from unhealthy ones. We expect other viral nanoparticles to form similar highly sensitive diagnostic assays for a variety of other protein markers.

Heterologous gene expression using self-assembled supra-molecules with high affinity for HSP70 chaperone

Contrary to the results of direct expression, various human proteins (ferritin light-chain, epithermal growth factor, interleukin-2, prepro-ghrelin, deletion mutants of glutamate decarboxylase and arginine deiminase, and mini-proinsulin) were all soluble in Escherichia coli cytoplasm when expressed with the N-terminus fusion of ferritin heavy-chain (FTN-H). Through systematic investigations, we have found that a specific peptide motif within FTN-H has a high affinity to HSP70 chaperone DnaK, and that the peptide motif was composed of a hydrophobic core of three residues (Ile, Phe and Leu) and two flanking regions enriched with polar residues (Gly, Gln and Arg). It was also observed that all the recombinant proteins expressed with the fusion of FTN-H formed spherical nanoparticles with diameters of 10–15 nm, as confirmed by the transmission electron microscopy image. The protein nanoparticles are non-covalently cross-linked supra-molecules formed by the self-assembly function of FTN-H. Upon the formation of the supra-molecule, its size is likely to be limited by the assembly properties of FTN-H, thereby keeping the self-assembled particles soluble. This study reports on the dual function of FTN-H for fusion expression and solubility enhancement of heterologous proteins: (i) high-affinity interaction with DnaK and (ii) formation of self-assembled supra-molecules with limited and constant sizes, thereby avoiding the undesirable formation of insoluble macro-aggregates of heterologous proteins.

The influence of glycosylation on secretion, stability, and immunogenicity of recombinant HBV pre-S antigen synthesized in Saccharomyces cerevisiae

Three types of recombinant pre-S antigens (i.e., pre-S1S2) of hepatitis B virus (HBV) were synthesized in Saccharomyces cerevisiae and secreted into extracellular medium: wild type (pre-S1S2) and two mutant antigens, pre-S1 degrees S2 (Asn15Gln) and pre-S1 degrees S2 degrees (Asn15Gln and Asn123Gln). An N-terminus sequence (Ser5-Ala28) of human interleukin 1 beta (hIL-1 beta) was used as synthetic prosequence of recombinant HBV surface antigen (pre-S), secreted from S. cerevisiae. The expression cassette comprised the signal peptide of the killer toxin of Kluyveromyces lactis, the synthetic prosequence above, KEX2 dibasic endopeptidase cleavage site (-Lys-Arg-), and the surface antigen. The recombinant pre-S1S2 and pre-S1 degrees S2 were secreted in the hyper-mannosylated form, while the recombinant pre-S1 degrees S2 degrees was produced without N-glycosylation. It has been demonstrated that the two particular N-linked glycans at Asn15 and Asn123 interfered with the B-cell response to the HBV-derived pre-S1S2, resulting in low titers of pre-S1S2-neutralizing antibodies. This problem was overcome by eliminating both of the N-glycosylation signals. Despite enhanced immunogenicity, the recombinant pre-S1 degrees S2 degrees showed two major problems: (1) inefficient Kex2 cleavage process in the secretory pathway and (2) the severe proteolytic degradation by yeast proteases. The efficiency of Kex2 cleavage increased dramatically by removing N-glycosylation signal in the synthetic prosequence, but the proteolysis of pre-S1 degrees S2 degrees was somewhat inevitable. Further systematic approaches including modulation of degree of N-glycosylation or relocation of N-glycosylation sites in the recombinant pre-S1S2 may make it possible to achieve both enhanced immunogenicity and resistance towards proteolytic degradation of the secreted pre-S antigen.

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 β‐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.—Lee S‐H., Lee, H., Park J‐S., Choi, H., Han K‐Y., Seo H‐S., Ahn K‐Y., Han S‐S., Cho, Y., Lee K‐H., Lee J. A novel approach to ultrasensitive diagnosis using supramolecular protein nano‐particles. FASEB J. 21, 1324–1334 (2007)

Proteomic response analysis of a threonine-overproducing mutant of Escherichia coli

The proteomic response of a threonine-overproducing mutant of Escherichia coli was quantitatively analysed by two-dimensional electrophoresis. Evidently, 12 metabolic enzymes that are involved in threonine biosynthesis showed a significant difference in intracellular protein level between the mutant and native strain. The level of malate dehydrogenase was more than 30-fold higher in the mutant strain, whereas the synthesis of citrate synthase seemed to be severely inhibited in the mutant. Therefore, in the mutant, it is probable that the conversion of oxaloacetate into citrate was severely inhibited, but the oxidation of malate to oxaloacetate was significantly up-regulated. Accumulation of oxaloacetate may direct the metabolic flow towards the biosynthetic route of aspartate, a key metabolic precursor of threonine. Synthesis of aspartase (aspartate ammonia-lyase) was significantly inhibited in the mutant strain, which might lead to the enhanced synthesis of threonine by avoiding unfavourable degradation of aspartate to fumarate and ammonia. Synthesis of threonine dehydrogenase (catalysing the degradation of threonine finally back to pyruvate) was also significantly down-regulated in the mutant. The far lower level of cystathionine beta-lyase synthesis in the mutant seems to result in the accumulation of homoserine, another key precursor of threonine. In the present study, we report that the accumulation of important threonine precursors, such as oxaloacetate, aspartate and homoserine, and the inhibition of the threonine degradation pathway played a critical role in increasing the threonine biosynthesis in the E. coli mutant.

A Novel Bioassay Platform Using Ferritin‐Based Nanoprobe Hydrogel

Ferritin-based nanoprobe (FBNP) hydrogel was synthesized through a simple one-step copolymerization and used as a diagnostic assay platform to solve the traditional problems [ 1–6 ] (i.e., low sensitivity and specifi city, infeasibility of multiplex assays, random orientation of probes, probe instability, uncontrollable probe loading, etc.) of ELISA (enzyme-linked immunosorbent assay)-based bioassays. Here we show the advantages of the diagnostic assays based on FBNP hydrogel: probe immobilization without a random orientation problem, controllable loading of homogeneously oriented probes, protein-friendly environment, suffi cient storage stability, much higher sensitivity than ELISA, and high specifi city and reproducibility even in multiplex assays. FBNP hydrogel was successfully applied to the sensitive and specifi c diagnostic assays of acquired immune defi ciency syndrome (AIDS) [ 7–9 ] and Sjögren’s syndrome (SS). [ 10–12 ] Although the diagnostic assays of AIDS and SS were demonstrated as proof-of-concept in this study, FBNP hydrogel can be applied in general to sensitive and specifi c detection of many other disease markers. Proteins are chemically and structurally complex with a heterogeneous outer surface and they easily lose their structure and biochemical activity as a result of denaturation, dehydration, or oxidation. Therefore, in protein detection assays that are based on specifi c interactions between the protein probe and protein analyte/target/marker (e.g., antibody–antigen interaction), it is of crucial importance to design a novel assay platform that stably maintains the native conformation, analyte binding affi nity, high surface density, and stability of welloriented protein probes on the assay surface. [ 13 , 14 ] The conventional methods that use protein probes are based on attachment of probes on the reactive surface that is modifi ed either for passive adsorption or for covalent coupling of primary amine

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

BackgroundThe 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.ResultsWe 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.ConclusionRpoS 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.

Fluorescent viral nanoparticles with stable in vitro and in vivo activity

We synthesized fluorescent capsid nanoparticles (FCNPs) by genetically inserting fluorescent protein (FP) (DsRed or eGFP) into each of 240 surface spike tips of hepatitis B virus (HBV) capsid particles. That is, when expressed in E. coli, FCNPs formed spherical nanoparticles with uniform diameter of about 40 nm owing to the self-assembly function of HBV core protein (i.e. basic assembly unit of capsid) and were successfully purified through Ni(+2) affinity- and sucrose gradient based purification. We also added the glycine-rich fexible linker peptides in between DsRed (or eGFP) and capsid to reduce fluorescence quenching among the densely displayed DsReds (or eGFPs) on the capsid surface. As compared to cognate fluorescent monomer proteins, it is notable that FCNPs showed a significantly amplified (160-170-fold) fluorescence intensity and enhanced conformational stability even in 50% serum solutin at 37 °C. The high conformational stability of FCNPs seems to result both from the highly stable structure of HBV capsid particles and from the well oriented insertion of fluorescent protein into capsid spike tip to keep native conformation of DsRed or eGFP. When estimated with continuous exposure to strong excitation light, FCNPs also showed much higher photostability than DsRed, eGFP, and a commonly used organic fluorescent dye, which happened presumably because the enhanced conformational stability of FCNPs significantly reduced photobleaching of fluorophores. Especially, it is notable that rFCNPs stably emitted high-level fluorescence inside mouse for a prolonged period, thereby showing high in vivo stability. The developed FCNPs are likely to have a great potential to be used as an effective and non-cytotoxic tool for in vivo optical imaging as well as in vitro fluorescent reporter in various biomolecular detection assays.

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.

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.