• Parasites, Hosts and Diseases
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• v.60 no.2
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• pp.143-147
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• 2022

Acanthamoeba keratitis (AK) is a rare ocular disease, but it is a painful and sight-threatening infectious disease. Early diagnosis and adequate treatment are necessary to prevent serious complications. While AK is frequently diagnosis via several PCR assays or Acanthamoeba-specific antibodies, a more specific and effective diagnostic method is required. This study described the production of a polyclonal peptide antibody against the periplasmic binding protein (PBP) of A. castellanii and investigated its diagnostic potential. Western blot analysis showed that the PBP antibody specifically reacted with the cell lysates of A. castellanii. However, the PBP antibody did not interact with human corneal epithelial (HCE) cells and the other 3 major causative agents of keratitis. Immunocytochemistry (ICC) results revealed the specific detection of A. castellanii trophozoites and cysts by PBP antibodies when A. castellanii were co-cultured with HCE cells. PBP antibody specificity was further confirmed by co-culture of A. castellanii trophozoites with F. solani, S. aureus, and P. aeruginosa via ICC. The PBP antibody specifically reacted with the trophozoites and cysts of A. polyphaga, A. hatchetti, A. culbertsoni, A. royreba, and A. healyi, thus demonstrated its genus-specific nature. These results showed that the PBP polyclonal peptide antibody of A. castellanii could specifically detect several species of Acanthamoeba, contributing to the development of an effective antibody-based AK diagnostics.

• Journal of Microbiology and Biotechnology
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• v.27 no.11
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• pp.1999-2009
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• 2017

The secretion efficiency of a protein in a Sec-type secretion system is mainly determined by an N-terminal signal peptide and its combination with its cognate protein. Five signal peptides, namely, two synthetic Sec-type and three Bacillus licheniformis alpha-amylase-derived signal peptides, were compared for periplasmic expression of the human growth hormone (hGH) in E. coli. Based on in silico predictions on the signal peptides' cleavage efficiencies and their corresponding mRNA secondary structures, a number of amino acid substitutions and silent mutations were considered in the modified signal sequences. The two synthetic signal peptides, specifically designed for hGH secretion in E. coli, differ in their N-terminal positively charged residues and hydrophobic region lengths. According to the mRNA secondary structure predictions, combinations of the protein and each of the five signal sequences could lead to different outcomes, especially when accessibility of the initiator ATG and ribosome binding sites were considered. In the experimental stage, the two synthetic signal peptides displayed complete processing and resulted in efficient secretion of the mature hGH in periplasmic regions, as was demonstrated by protein analysis. The three alpha-amylase-derived signal peptides, however, were processed partially from their precursors. Therefore, to achieve efficient secretion of a protein in a heterologous system, designing a specific signal peptide by using a combined approach of optimizations of the mRNA secondary structure and the signal peptide H-domain and cleavage site is recommended.

• Microbiology and Biotechnology Letters
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• v.49 no.2
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• pp.148-156
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• 2021

Single-chain antibodies against epidermal growth factor receptor variant III (EGFRvIII) are potentially promising agents for developing antibody-based cancer treatment strategies. We described in our previous study the successful expression of an anti-EGFRvIII scFv antibody in Escherichia coli. However, we could also observe the formation of insoluble aggregates in the periplasmic space, limiting the production yield of the active product. In the present study, we investigated the mechanisms by which growth conditions could affect the expression of the soluble anti-EGFRvIII scFv antibody in small-scale E. coli NiCo21(DE3) cultures, attempting to maximize production. The secreted scFv molecules were purified using Ni-NTA magnetic beads and protein characterization was performed using SDS-PAGE and western blot analyses. We used the ImageJ software for protein quantification and determined the antigen-binding activity of the scFv antibody against the EGFRvIII protein. Our results showed that the highest percentage of soluble scFv expression could be achieved under culture conditions that combined low IPTG concentration (0.1 mM), low growth temperature (18℃), and large culture dish surface area. We found moderate-yield soluble scFv production in the culture medium after lactose-mediated induction, which was also beneficial for downstream protein processing. These findings were confirmed by conducting western blot analysis, indicating that the soluble, approximately 30-kDa scFv molecule was localized in the periplasm and the extracellular space. Moreover, the antigen-binding assay confirmed the scFv affinity against the EGFRvIII antigen. In conclusion, our study reveals that low-speed protein expression is preferable to obtain more soluble anti-EGFRvIII scFv protein in an E. coli expression system.

• Microbiology and Biotechnology Letters
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• v.34 no.1
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• pp.15-22
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• 2006

A 4,971 bp chromosomal DNA fragment containing the pqrA, paraquat resistance gene, was cloned from Ochrobactrum anthropi JW-2, and the complete nucleotide sequence was determined. Nucleotide and deduced amino acid sequences of the fragment revealed the presence of 4 complete ORFs (orf2, pqrA, orf3, orf4) and two incomplete ORFs(orf1, orf5). Orf1, pqrA, orf4 and orf5 exists at the direct strand but orf2 and orf3 exists at the reverse complementary strand. Orf1 which of incomplete sequences without start codon shares homology with ATP binding region of the response regulator receiver. Orf2 shares high homology with members of the tetR family of transcriptional repressor which have a helix-turn-helix (H-T-H) motif. Therefore, the orf2 is predicted as a transcriptional repressor of pqrA and is designated as pqrR2. Orf3 shares high homology with the members of the lysR family acting as a transcriptional activator which have both of a H-T-H motif at the N-terminal region and substrate binding domain at the C-terminal region. Therefore, the orf3 is predicted as a transcriptional activator of pqrA and is designated as pqrR1. Orf4 shows homology with the periplasmic substrate-binding protein of amino acid ABC transporter. Orf5 which of incomplete sequences without stop codon revealed the homology with the permeases protein of amino acid ABC transporter.

• Journal of Life Science
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• v.28 no.12
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• pp.1416-1423
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• 2018

With strong biotin binding affinity ($K_D=10^{-14}M$), the tetrameric feature of streptavidin could be used to increase the antigen binding activity of a camel heavy chain (VHH) antibody through their fusion, here stained with biotinylated horseradish peroxidase and subsequent immunoassays ELISA and Western blot analysis. For this application, we cloned the streptavidin gene amplified from the Streptomyces avidinii chromosome by PCR, and this was fused to the gene of the 8B9 VHH antibody which is specific to green fluorescent protein (GFP) antigens. To express a soluble fusion protein in Escherichia coli, we used the pUC119 plasmid-based expression system which uses the lacZ promoter for induction by IPTG, the pelB leader sequence at the N-terminus for secretion into the periplasmic space, and six polyhistidine tags at the C-terminus for purification of the expressed proteins using an $Ni^+$-NTA-agarose column. Although streptavidin is toxic to E. coli because of its strong biotin binding property, this soluble fusion protein was expressed successfully. In SDS-PAGE, the size of the purified fusion protein was 122.4 kDa in its native condition and 30.6 kDa once denatured by boiling, suggesting the tetramerization of the monomeric subunit by non-covalent association through the streptavidin moiety fusing to the 8B9 VHH antibody. In addition, this fusion protein showed biotin binding activity similar to streptavidin as well as GFP antigen binding activity through both ELISA and Western blot analysis. In conclusion, the protein resulting from the fusion of an 8B9 VHH antibody with streptavidin was successfully expressed and purified as a soluble tetramer in E. coli; it showed both biotin and GFP antigen binding activity suggesting the possible production of a tetrameric and bifunctional VHH antibody.

• Molecules and Cells
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• v.37 no.2
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• pp.100-108
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• 2014

Salmonella enterica serovar Typhimurium (S. Typhimurium) is an intracellular pathogen that has evolved to survive in the phagosome of macrophages. The periplasmic copper-binding protein CueP was initially known to confer copper resistance to S. Typhimurium. Crystal structure and biochemical studies on CueP revealed a putative copper binding site surrounded by the conserved cysteine and histidine residues. A recent study reported that CueP supplies copper ions to periplasmic Cu,Zn-superoxide dismutase (SodCII) at a low copper concentration and thus enables the sustained SodCII activity in the periplasm. In this study, we investigated the role of CueP in copper resistance at a high copper concentration. We observed that the survival of a cueP-deleted strain of Salmonella in macrophage phagosome was significantly reduced. Subsequent biochemical experiments revealed that CueP specifically mediates the reduction of copper ion using electrons released during the formation of the disulfide bond. We observed that the copper ion-mediated Fenton reaction in the presence of hydrogen peroxide was blocked by CueP. This study provides insight into how CueP confers copper resistance to S. Typhimurium in copper-rich environments such as the phagosome of macrophages.

• BMB Reports
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• v.38 no.3
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• pp.266-274
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• 2005

High temperature requirement A (HtrA) and its homologues constitute the HtrA familiy proteins, a group of heat shock-induced serine proteases. Bacterial HtrA proteins perform crucial functions with regard to protein quality control in the periplasmic space, functioning as both molecular chaperones and proteases. In contrast to other bacterial quality control proteins, including ClpXP, ClpAP, and HslUV, HtrA proteins contain no regulatory components or ATP binding domains. Thus, they are commonly referred to as ATP-independent chaperone proteases. Whereas the function of ATP-dependent chaperone-proteases is regulated by ATP hydrolysis, HtrA exhibits a PDZ domain and a temperature-dependent switch mechanism, which effects the change in its function from molecular chaperone to protease. This mechanism is also related to substrate recognition and the fine control of its function. Structural and biochemical analyses of the three HtrA proteins, DegP, DegQ, and DegS, have provided us with clues as to the functional regulation of HtrA proteins, as well as their roles in protein quality control at atomic scales. The objective of this brief review is to discuss some of the recent studies which have been conducted regarding the structure and function of these HtrA proteins, and to compare their roles in the context of protein quality control.

• Journal of Life Science
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• v.25 no.6
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• pp.631-637
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• 2015

Streptavidin, a protein produced by Streptomyces avidinii, strongly binds up to four molecules of vitamin H, d-biotin exhibiting the dissociation constant of about 10⁻¹⁵ M. This strong binding affinity has been applied for detection and characterization of numerous biological molecules suggesting expression and purification of functional streptavidin should be very useful for the application of this streptavidin-biotin interaction. To express a soluble streptavidin in Escherichia coli, We synthesized streptavidin genes and cloned into pET-22b plasmid, which uses T7 RNA polymerase/T7 promoter expression systems containing pelB leader for secretion into periplasmic space and six polyhistidine tags at C-terminus for purification of expressed proteins. Although streptavidin is toxic to Escherichia coli due to strong biotin binding property, streptavidin was expressed very sufficiently in a range of 10-20 mg/ml. In SDS-PAGE, the size of purified protein was shown as 17 kDa in denatured condition (boiling) and 68 kDa in native condition (without boiling) suggesting tetramerization of monomeric subunit by non-covalent association. Further analysis by size-exclusion chromatography supported streptavidin’s tetrameric structure as well. In addition, soluble streptavidin detected biotinylated proteins in westernblot indicating its functional activity to biotin. Taken these results together, it concluded that our simple expression system was able to show high yield, homotetrameric formation and biotin binding activity analogous to natural streptavidin.

• Journal of Microbiology and Biotechnology
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• v.22 no.9
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• pp.1271-1278
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• 2012

Genetic fusion of two proteins frequently induces beneficial effects to the proteins, such as increased solubility, besides the combination of two protein functions. Here, we study the effects of the bacterial surface layer protein SgsE from Geobacillus stearothermophilus NRS 2004/3a on the folding of a C-terminally fused enhanced green fluorescent protein (EGFP) moiety. Although GFPs are generally unable to adopt a functional confirmation in the bacterial periplasm of Escherichia coli cells, we observed periplasmic fluorescence from a chimera of a 150-amino-acid N-terminal truncation of SgsE and EGFP. Based on this finding, unfolding and refolding kinetics of different S-layer-EGFP chimeras, a maltose binding protein-EGFP chimera, and sole EGFP were monitored using green fluorescence as indicator for the folded protein state. Calculated apparent rate constants for unfolding and refolding indicated different folding pathways for EGFP depending on the fusion partner used, and a clearly stabilizing effect was observed for the SgsE_C fusion moiety. Thermal stability, as determined by differential scanning calorimetry, and unfolding equilibria were found to be independent of the fused partner. We conclude that the stabilizing effect SgsE_C exerts on EGFP is due to a reduction of degrees of freedom for folding of EGFP in the fused state.

• Parasites, Hosts and Diseases
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• v.56 no.6
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• pp.553-558
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• 2018

Pathogenic Acanthamoeba spp. cause granulomatous amoebic encephalitis and keratitis. Acanthamoeba keratitis (AK) is a rare but serious ocular infection that can result in permanent visual impairment or blindness. However, pathogenic factors of AK remain unclear and treatment for AK is arduous. Expression levels of proteins secreted into extracellular space were compared between A. castellanii pathogenic (ACP) and non-pathogenic strains. Two-dimensional polyacrylamide gel electrophoresis revealed 123 differentially expressed proteins, including 34 increased proteins, 7 qualitative increased proteins, 65 decreased proteins, and 17 qualitative decreased proteins in ACP strain. Twenty protein spots with greater than 5-fold increase in ACP strain were analyzed by liquid chromatography triple quadrupole mass spectrometry. These proteins showed similarity each to inosine-uridine preferring nucleoside hydrolase, carboxylesterase, oxygen-dependent choline dehydrogenase, periplasmic-binding protein proteinases and hypothetical proteins. These proteins expressed higher in ACP may provide some information to understand pathogenicity of Acanthamoeba.