Kyung Yeon Han

Sensitive and simultaneous detection of cardiac markers in human serum using surface acoustic wave immunosensor.

We present a rapid and sensitive surface acoustic wave (SAW) immunosensor that utilizes gold staining as a signal enhancement method. A sandwich immunoassay was performed on sensing area of the SAW sensor, which could specifically capture and detect cardiac markers (cardiac troponin I (cTnI), creatine kinase (CK)-MB, and myoglobin). The analytes in human serum were captured on gold nanoparticles (AuNPs) that were conjugated in advance with detection antibodies. Introduction of these complexes to the capture antibody-immobilized sensor surface resulted in a classic AuNP-based sandwich immunoassay format that has been used for signal amplification. In order to achieve further signal enhancement, a gold staining method was performed, which demonstrated that it is possible to obtain gold staining-mediated signal augmentation on a mass-sensitive device. The sensor response due to gold staining varied as a function of cardiac marker concentration. We also investigated effects of increasing operating frequency on sensor responses. Results showed that detection limit of the SAW sensor could be further improved by increasing the operating frequency.

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.

Proteome response of Escherichia coli fed-batch culture to temperature downshift

During fed-batch cultivation of Escherichia coli K-12, the proteomic response to a temperature downshift from 37 to 20°C was quantitatively monitored and analyzed by using two-dimensional electrophoresis. When the temperature of exponentially growing E. coli K-12 culture was downshifted to 20°C, the synthesis level of 57 intracellular proteins showed significant changes for a prolonged period of time, compared to the fed-batch culture controlled at 37°C. Thus, these proteins are regarded as important stress proteins responsive to cold shock, which were analyzed by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and identified using the E. coli SWISS-2DPAGE database. Most of the identified proteins were shown to be involved in energy metabolism, several cellular molecule biosynthetic pathways and catabolism, cell processes, flagellar biosynthesis and motility, and protein translation and folding. The systematic approach to the monitoring of proteomic responses and the detailed analysis results reported in this article would be useful in understanding the metabolic adaptation to lowered culture temperature and designing efficient fermentation strategies for the production of recombinant proteins and metabolites using E. coli strains.

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)

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.

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.

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

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

Escherichia coli EDA is a novel fusion expression partner to improve solubility of aggregation-prone heterologous proteins.

Since the use of solubility enhancer proteins is one of the effective methods to produce active recombinant proteins within Escherichia coli, the development of a novel fusion expression partner that can be applied to various aggregation-prone proteins is of crucial importance. In our previous work, two-dimensional electrophoresis (2-DE) was employed to systematically analyze the E. coli BL21 (DE3) proteome profile in response to heat treatment, and KDPG aldolase (EDA) was identified as a heat-responsive and aggregation-resistant protein. When used as fusion expression partner, EDA significantly increased the solubility of seven aggregation-prone heterologous proteins in the E. coli cytoplasm. The efficacy of EDA as a fusion expression partner was evaluated through the analysis of bioactivity or secondary structure of several target proteins: EDA-fusion expression resulted in the synthesis of bioactive human ferritin light chain and bacterial arginine deiminase and the formation of correct secondary structure of human granulocyte colony stimulation factor.

The N-domain of Escherichia coli phosphoglycerate kinase is a novel fusion partner to express aggregation-prone heterologous proteins†

As a fusion partner to express aggregation‐prone heterologous proteins, we investigated the efficacy of Escherichia coli phosphoglycerate kinase (ePGK) that consists of two functional domains (N‐ and C‐domain) and reportedly has a high structural stability. When the full‐length ePGK (F‐ePGK) was used as a fusion partner, the solubility of the heterologous proteins increased, but some of them still had a large fraction of insoluble aggregates. Surprisingly, the fusion expression using the N‐domain of ePGK (N‐ePGK) made the insoluble fraction significantly reduce to less than 10% for all the heterologous fusion proteins tested. Also, we evaluated the efficacy of N‐ePGK in making the target proteins be expressed with their own native function or structure. It was found that of human ferritin light chain, bacterial arginine deiminase, human granulocyte colony stimulating factor were synthesized evidently with the self‐assembly function, L‐arginine‐degrading activity, and the correct secondary structure, respectively, through the fusion expression using N‐ePGK. These results indicate that N‐ePGK is a highly potent fusion partner that can be widely used for the synthesis of a variety of heterologous proteins in E. coli. Biotechnol. Bioeng. 2012; 109:325–335.