Journal of Microbiology

Journal Article

Comparative Secretory Efficiency of Two Chitosanase Signal Peptides from Bacillus subtilis in Escherichia coli: Tae-Yang Eom, Yehui Gang, Youngdeuk Lee, Yoon-Hyeok Kang, Eunyoung Jo, Svini Dileepa Marasinghe, Heung Sik Park, Gun-Hoo Park, Chulhong Oh; J. Microbiol. 2024;62(12):1155-1164. Published online November 25, 2024; DOI: https://doi.org/10.1007/s12275-024-00186-1

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Abstract: The production of recombinant proteins in Escherichia coli is often challenged by cytoplasmic expression due to proteolytic degradation and inclusion body formation. Extracellular expression can overcome these problems by simplifying downstream processing and improving protein yields. This study aims to compare the efficiency of two Bacillus subtilis chitosanase signal peptides in mediating extracellular secretion in E. coli. We identified a naturally occurring mutant signal peptide (mCsn2-SP) from B. subtilis CH2 chitosanase (CH2CSN), which is characterized by a deletion of six amino acids in the N-region relative to the signal peptide (Csn1-SP) from B. subtilis CH1 chitosanase (CH1CSN). The CH1CSN and CH2CSN genes were cloned into the pET-11a vector and protein secretion was evaluated in E. coli BL21(DE3) host cells. Expression was induced with 0.1 mM and 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 30 °C for one and three days. CH2CSN showed higher secretion levels compared to CH1CSN under all experimental conditions, especially with 0.1 mM IPTG induction for 3 days, which resulted in a 2.37-fold increase in secretion. Furthermore, it was demonstrated that mCsn2-SP is capable of secreting human Cu,Zn-superoxide dismutase (hSOD) in E. coli BL21(DE3) and successfully translocating it to the periplasmic region. This study represents the inaugural investigation into the utilisation of a naturally modified signal peptide, thereby corroborating the assertion that signal peptide deletion variants can influence protein secretion efficiency. Furthermore, the findings substantiate the proposition that such variants can serve as a viable alternative for the secretion of heterologous proteins in E. coli.

Reviews

Ammonia-oxidizing archaea in biological interactions: Jong-Geol Kim , Khaled S. Gazi , Samuel Imisi Awala , Man-Young Jung , Sung-Keun Rhee; J. Microbiol. 2021;59(3):298-310. Published online February 23, 2021; DOI: https://doi.org/10.1007/s12275-021-1005-z

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The third domain Archaea was known to thrive in extreme or anoxic environments based on cultivation studies. Recent metagenomics- based approaches revealed a widespread abundance of archaea, including ammonia-oxidizing archaea (AOA) of Thaumarchaeota in non-extreme and oxic environments. AOA alter nitrogen species availability by mediating the first step of chemolithoautotrophic nitrification, ammonia oxidation to nitrite, and are important primary producers in ecosystems, which affects the distribution and activity of other organisms in ecosystems. Thus, information on the interactions of AOA with other cohabiting organisms is a crucial element in understanding nitrogen and carbon cycles in ecosystems as well as the functioning of whole ecosystems. AOA are self-nourishing, and thus interactions of AOA with other organisms can often be indirect and broad. Besides, there are possibilities of specific and obligate interactions. Mechanisms of interaction are often not clearly identified but only inferred due to limited knowledge on the interaction factors analyzed by current technologies. Here, we overviewed different types of AOA interactions with other cohabiting organisms, which contribute to understanding AOA functions in ecosystems.

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Nitrogen cycling process and application in different prawn culture modes
Zhao Chen, Jian Li, Qianqian Zhai, Zhiqiang Chang, Jitao Li
Reviews in Aquaculture.2024; 16(4): 1580. CrossRef
Multidrug-resistant plasmid RP4 inhibits the nitrogen removal capacity of ammonia-oxidizing archaea, ammonia-oxidizing bacteria, and comammox in activated sludge
Zhaohui Zhang, Lin Bo, Shang Wang, Chenyu Li, Xi Zhang, Bin Xue, Xiaobo Yang, Xinxin He, Zhiqiang Shen, Zhigang Qiu, Chen Zhao, Jingfeng Wang
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MINIREVIEW] Advances in novel influenza vaccines: a patent review: Jae-Min Song; J. Microbiol. 2016;54(6):403-412. Published online May 27, 2016; DOI: https://doi.org/10.1007/s12275-016-6176-7

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Abstract

The threat of a major human influenza pandemic such as the avian H5N1 or the 2009 new H1N1 has emphasized the need for effective prevention strategies to combat these pathogens. Although egg based influenza vaccines have been well established for a long time, it remains an ongoing public health need to develop alternative production methods that ensures improved safety, efficacy, and ease of administration compared with conventional influenza vaccines. This article is intended to cover some of the recent advances and related patents on the development of influenza vaccines including live attenuated, cell based, genomic and synthetic peptide vaccines.

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Plant-made virus-like particles bearing influenza hemagglutinin (HA) recapitulate early interactions of native influenza virions with human monocytes/macrophages
Alexander I. Makarkov, Sabrina Chierzi, Stéphane Pillet, Keith K. Murai, Nathalie Landry, Brian J. Ward
Vaccine.2017; 35(35): 4629. CrossRef

Research Support, Non-U.S. Gov't

Low-Scale Expression and Purification of an Active Putative Iduronate 2-Sulfate Sulfatase-Like Enzyme from Escherichia coli K12: Edwin David Morales-Álvarez , Claudia Marcela Rivera-Hoyos , Angélica María Baena-Moncada , Patricia Landázuri , Raúl A. Poutou-Piñales , Homero Sáenz-Suárez , Luis A. Barrera , Olga Y. Echeverri-Peña; J. Microbiol. 2013;51(2):213-221. Published online April 27, 2013; DOI: https://doi.org/10.1007/s12275-013-2416-2

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15 Scopus

Abstract: The sulfatase family involves a group of enzymes with a large degree of similarity. Until now, sixteen human sulfatases have been identified, most of them found in lysosomes. Human deficiency of sulfatases generates various genetic disorders characterized by abnormal accumulation of sulfated intermediate compounds. Mucopolysaccharidosis type II is characterized by the deficiency of iduronate 2-sulfate sulfatase (IDS), causing the lysosomal accumulation of heparan and dermatan sulfates. Currently, there are several cases of genetic diseases treated with enzyme replacement therapy, which have generated a great interest in the development of systems for recombinant protein expression. In this work we expressed the human recombinant IDS-Like enzyme (hrIDS-Like) in Escherichia coli DH5α. The enzyme concentration revealed by ELISA varied from 78.13 to 94.35 ng/ml and the specific activity varied from 34.20 to 25.97 nmol/h/mg. Western blotting done after affinity chromatography purification showed a single band of approximately 40 kDa, which was recognized by an IgY polyclonal antibody that was developed against the specific peptide of the native protein. Our 100 ml-shake-flask assays allowed us to improve the enzyme activity seven fold, compared to the E. coli JM109/pUC13-hrIDS-Like system. Additionally, the results obtained in the present study were equal to those obtained with the Pichia pastoris GS1115/ pPIC-9-hrIDS-Like system (3 L bioreactor scale). The system used in this work (E. coli DH5α/pGEX-3X-hrIDS-Like) emerges as a strategy for improving protein expression and purification, aimed at recombinant protein chemical characterization, future laboratory assays for enzyme replacement therapy, and as new evidence of active putative sulfatase production in E. coli.

Retracted Publication

Identification and Characterization of a Novel Antibacterial Peptide, Avian β-Defensin 2 from Ducks: Deying Ma , Ruiqin Wang , Wenyan Liao , Zongxi Han , Shengwang Liu; J. Microbiol. 2009;47(5):610-618. Published online October 24, 2009; DOI: https://doi.org/10.1007/s12275-009-0068-z

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33 Scopus

Abstract: In this study, a novel avian β-defensin (AvBD) was isolated from duck pancreas. The complete nucleotide sequence of the gene contained an 195 bp open reading frame encoding 64 amino acids. Homology, characterization and comparison of the gene with AvBD from other avian species confirmed that it was duck AvBD2. The mRNA expression of the gene was analyzed in 17 tissues from 21-day-old ducks. AvBD2 was highly expressed in the trachea, crop, heart, bone marrow, and pancreas; moderately expressed in the muscular stomach, small intestine, kidney, spleen, thymus, and bursa of Fabricius; and weakly expressed in skin. We produced and purified recombinant AvBD2 by expressing the gene in Escherichia coli. As expected, the recombinant peptide exhibited strong bactericidal properties against Bacillus cereus, Staphylococcus aureus, and Pasteurella multocida, and weak bactericidal properties against E. coli and Salmonella choleraesuis. In addition, the recombinant protein retained antimicrobial activity against S. aureus under different temperatures (range, -20°C to 100°C) and pH values (range, 3 to 12).

Research Support, Non-U.S. Gov't

Stable Expression and Secretion of Polyhydroxybutyrate Depolymerase of Paucimonas lemoignei in Escherichia coli: Se Whan Park , Moon Gyu Chung , Hwa Young Lee , Jeong Yoon Kim , Young Ha Rhee; J. Microbiol. 2008;46(6):662-669. Published online December 24, 2008; DOI: https://doi.org/10.1007/s12275-008-0283-z

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Abstract: An efficient strategy for the expression and secretion of extracellular polyhydroxybutyrate depolymerase (PhaZ1) of Paucimonas lemoignei in Escherichia coli was developed by employing the signal peptide of PhaZ1 and a truncated ice nucleation protein anchoring motif (INPNC). Directly synthesized mature form of PhaZ1 was present in the cytoplasm of host cells as inclusion bodies, while a construct containing PhaZ1 and its own N-terminal signal peptide (PrePhaZ1) enabled the secretion of active PhaZ1 into the extracellular medium. However, the PrePhaZ1 construct was harmful to the host cell and resulted in atypical growth and instability of the plasmid during the cultivation. In contrast, INPNC-PhaZ1 and INPNCPrePhaZ1 fusion constructs did not affect growth of host cells. INPNC-PhaZ1 was successfully displayed on the cell surface with its fusion form, but did not retain PhaZ1 activity. In the case of INPNC-PrePhaZ1, the initially synthesized fusion form was separated by precise cleavage of the signal peptide, and active PhaZ1 was consequently released into the culture medium. The amount of PhaZ1 derived from E. coli (INPNC-PrePhaZ1) was almost twice as great as that directly expressed from E. coli (PrePhaZ1), and was predominantly (approximately 85%) located in the periplasm when cultivated at 22°C but was efficiently secreted into the extracellular medium when cultivated at 37°C.

Journal Article

Biologically Active and C-Amidated HinnavinII-38-Asn Produced from a Trx Fusion Construct in Escherichia coli: Chang Soo Kang , Seung-Yeol Son , In Seok Bang; J. Microbiol. 2008;46(6):656-661. Published online December 24, 2008; DOI: https://doi.org/10.1007/s12275-008-0214-z

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9 Scopus

Abstract: The cabbage butterfly (Artogeia rapae) antimicrobial peptide hinnavinII as a member of cecropin family is synthesized as 37 residues in size with an amidated lysine at C-terminus and shows the humoral immune response to a bacterial invasion. In this work, a synthetic gene for hinnavinII-38-Asn (HIN) with an additional amino acid asparagine residue containing amide group at C-terminus was cloned into pET-32a(+) vector to allow expression of HIN as a Trx fusion protein in Escherichia coli strain BL21 (DE3) pLysS. The resulting expression level of the fusion protein Trx-HIN could reach 15~20% of the total cell proteins and more than 70% of the target proteins were in soluble form. The fusion protein could be purified successfully by HiTrap Chelating HP column and a high yield of 15 mg purified fusion protein was obtained from 80 ml E. coli culture. Recombinant HIN was readily obtained by enterokinase cleavage of the fusion protein followed by FPLC chromatography, and 3.18 mg pure active recombinant HIN was obtained from 80 ml culture. The molecular mass of recombinant HIN determined by MALDI-TOF mass spectrometer is 4252.084 Da which matches the theoretical mass (4252.0 Da) of HIN. Comparing the antimicrobial activities of the recombinant hinnavinII with C-amidated terminus to that without an amidated C-terminus, we found that the amide of asparagine at C-terminus of hinnavinII improved its potency on certain microorganism such as E. coli, Enterobacter cloacae, Bacillus megaterium, and Staphylococcus aureus.

Research Support, Non-U.S. Gov'ts

Development of a Simple Cell Lysis Method for Recombinant DNA Using Bacteriophage Lambda Lysis Genes: Boyun Jang , Yuna Jung , Dongbin Lim; J. Microbiol. 2007;45(6):593-596.; DOI: https://doi.org/2602 [pii]

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Abstract: In this study, we describe the development of a simple and efficient method for cell lysis via the insertion of a bacteriophage lambda lysis gene cluster into the pET22b expression vector in the following order; the T7 promoter, a gene for a target protein intended for production, Sam7 and R. This insertion of R and Sam7 into pET22b exerted no detrimental effects on cellular growth or the production of a target protein. The induction of the T7 promoter did not in itself result in the autolysis of cells in culture but the harvested cells were readily broken by freezing and thawing. We compared the efficiency of the cell lysis technique by freezing and thawing to that observed with sonication, and determined that both methods completely disintegrated the cells and released proteins into the solution. With our modification of pET22b, the lysis of cells became quite simple, efficient, and reliable. This strategy may prove useful for a broad variety of applications, particularly in experiments requiring extensive cell breakage, including library screening and culture condition exploration, in addition to protein purification.

Characterization of Recombinant Drosophila melanogaster Myo-inositol-1-phosphate Synthase Expressed in Escherichia coli: Sang-Hee Park , Jong-Il Kim; J. Microbiol. 2004;42(1):20-24.; DOI: https://doi.org/2006 [pii]

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Abstract: Cloned myo-inositol-1-phpsphate synthase (INOS) of Drosophila melanogaster was expressed in Escherichia coli, and purified using a His affinity column. The purified INOS required NAD^+ for the conversion of glucose-6-phosphate to inositol-1-phosphate. The optimum pH for myo-inositol-1-phosphate synthase is 7.5, and the maximum activity was measured at 40^oC. The molecular weight of the native enzyme, as determined by gel filtration, was approximately M_r 271,000±15,000. A single subunit of approximately M_r 62,000±5,000 was detected upon SDS-polyacrylamide gel electrophoresis. The Michaelis (K_m) and dissociation constants for glucose-6-phosphate were 3.5 and 3.7 mM, whereas for the cofactor NAD^+ these were 0.42 and 0.4 mM, respectively.

Expression of immunologically active porcine recombinant TGF-β1 precursor protein in baculovirus system: Lim, Hyun , Kim, Pyeung Hyeun , Chun, Gie Taek , Choi, Eui Yul , Yie, Se Won; J. Microbiol. 1997;35(4):341-346.

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Abstract: In order to express recombinant porcine TGF-β1 protein in a baculovirus expression system the entire TGF-β1 gene containing extra amino acids at the N terminus was cloned into pFBa and pFBb of the Bac-To-Bac^TM baculovirus expression system. One of the clones contained 106 extra amino acids and was designated pFBa-106 TGF-β1, and the other had 28 extra amino acids and was designated pFBb-28 TGF-β1. The orientation of the gene was identified with restriction enzyme mapping and PCR with internal TGF-β1 primers. Sf-9 cells were infected at a m.o.i. of 10 by the recombinant viruses generated from the two expected sizes of 55 kD and 46.4kD. these precursor forms of TGF-β1 with a polyclonal antibody against human TGF-β1. No mature form of TGF-β1 protein was detected on SDS gels and an immunoblot indicated that TGF-β1 precursor is not properlu processed in insect cells.

A Recombinant Mouse GM-CSF Protein Expressed as an Inclusion Form Shows Colony Stimulating Activity: Jin-Kyoo Kim , Eun-Jung Sohn , Soo-O Lee , Choon-Taek Lee , Ah Young Lee , Hye Kyung Chung , Bong Whan Sung , Hyun Joo Youn; J. Microbiol. 2000;38(2):109-112.

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Abstract: Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic hematopoietic growth factor and an activator of mature myeloid cells, and recombinant GM-CSF is increasingly under clinical studies for the treatment of various diseases including cancer, infectious diseases and hematopoietic diseases. We constructed a recombinant mouse GM-CSF expression plasmid with pelB leader sequence and His.Tag under T7 promoter control, and showed that the construct produced a 20 kDa recombinant protein in 8M urea. We also showed that the 20 kDa recombinant protein prepared in 8M urea stimulated colony formation in vitro, indicating that the recombinant mGM-CSF can be renatured to its native form to show the colony stimulating activity.

A Recombinant Human GM-CSF Protein Expressed as an Inclusion form in Escherichia coli Stimulates Colony Formation and Cell Proliferation in vitro: Ah Young Lee , Jin-Kyoo Kim , Hye Kyung Chung , Eun Kyong Bae , Jung Suk Hwang , Chung Won Cho , Dong Seok Lee , Jae Yong Han , Choon-Taek Lee , Soon-; J. Microbiol. 2002;40(1):77-81.

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Abstract: Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic hematopoietic growth factor involved in the development of myeloid cells from bone marrow, and an activator of mature myeloid cells functioning in a variety of antimicrobial and inflammatory responses. Recently, recombinant GM-CSF is increasingly under clinical study for treatment of various diseases including cancer, infectious diseases and hematopoietic diseases as well as for an immune response modulator. In this study, we constructed a recombinant human GM-CSF (rhGM-CSF) expression plasmid with a pelB leader sequence and His.Tag under T7 promoter control. The expression construct was shown to produce a recombinant protein of 20 kDa in the 8M urea preparation, indicating the rhGM-CSF may be expressed as an insoluble inclusion form. The 20 kDa recombinant protein in 8M urea was transformed into the water-soluble form by dialysis against PBS buffer (phosphate buffered saline). The soluble rhGM-CSF protein was shown to stimulate colony formation and cell proliferation in vitro, indicating that the rhGM-CSF could be refolded into its native form to show colony stimulating activity.

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