Jong Am Song

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.

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.

Human G-CSF synthesis using stress-responsive bacterial proteins

Abstract 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.

Synthesis of Mycoplasma arginine deiminase in E. coli using stress-responsive proteins

We found Escherichia coli proteins, elongation factor Ts (Tsf), and malate dehydrogenase (Mdh) that can exist in the form of native and soluble proteins even under stress situation such as heat shock and protein denaturing condition. To examine their property as solubility enhancers, aggregation-prone Mycoplasma arginine deiminase (mADI), which has been suggested as anti-cancer agent, was fused to the C-terminal of each of them and cloned into pET28a to be expressed in the E. coli cytoplasm. When mADI was fused to fusion partners (Mdh, Tsf), a significant portion of the recombinant mADI was expressed in a soluble fraction (>90%) whereas the directly expressed mADI was aggregated to the inclusion body. In addition, recombinant mADI released from the fusion tag retained its soluble form and presented its specific enzymatic activity of converting l-arginine into citrulline (>10 U/mg). These results show that Tsf and Mdh could serve as effective solubility enhancers for aggregation-prone proteins (e.g. mADI in this case) when used as fusion expression partners in bacterial expression systems.

A stress-responsive Escherichia coli protein, CysQ is a highly effective solubility enhancer for aggregation-prone heterologous proteins

When used as an N-terminal fusion expression partner, the Escherichia coli stress-responsive protein, CysQ dramatically increased the cytoplasmic solubility of various aggregation-prone heterologous proteins: Pseudomonas putida cutinase (CUT), human granulocyte colony-stimulating factor (hG-CSF), human ferritin light chain (hFTN-L), arginine deiminase (ADI), human interleukin-2 (IL2), human activation induced cytidine deaminase (AID), and deletion mutant of human glutamate decarboxylase (GAD448-585). As compared with well-known fusion tags such as glutathione-S-transferase (GST) and maltose-binding protein (MBP), the performance of CysQ as solubility enhancer was evidently better than GST and was similar to or better than MBP for the seven heterologous proteins above. This is likely due to the intrinsic ability of CysQ to form its native conformation, probably promoting the binding of molecular chaperones during the folding of CysQ-fusion protein. When used as a substrate, p-nitrophenyl butyrate (PNB) was successfully hydrolyzed to p-nitrophenol by CysQ-CUT fusion mutant. Even after CysQ was removed, the solubility of hFTN-L and hG-CSF, the secondary structure of hG-CSF, and self-assembly activity of hFTN-L were successfully maintained. Conclusively, it seems that CysQ is a highly effective solubility enhancer and fusion expression partner for the production of a variety of bio-active recombinant proteins.

YrhB is a highly stable small protein with unique chaperone-like activity in Escherichia coli BL21(DE3)

Escherichia coli YrhB (10.6 kDa) from strain BL21(DE3) that is commonly used for protein overexpression is a stable chaperone‐like protein and indispensable for supporting the growth of BL21(DE3) at 48 °C but not defined as conventional heat shock protein (HSP). YrhB effectively prevented heat‐induced aggregation of ribonucleotide synthetase (PurK). Without ATP, YrhB alone promoted in vitro refolding of uridine phosphorylase (UDP) and protected thermal denaturation of the refolded UDP. As a cis‐acting fusion partner, YrhB also significantly reduced inclusion body formation of nine aggregation‐prone heterologous proteins in BL21(DE3). Unlike conventional small HSPs, YrhB remained monomer under heat shock condition.