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.

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In silico analysis and experimental validation of lipoprotein and novel Tat signal peptides processing in Anabaena sp. PCC7120: Sonika Kumari , Akhilesh Kumar Chaurasia; J. Microbiol. 2015;53(12):837-846. Published online December 2, 2015; DOI: https://doi.org/10.1007/s12275-015-5281-3

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Signal peptide (SP) plays a pivotal role in protein translocation. Lipoprotein- and twin arginine translocase (Tat) dependent signal peptides were studied in All3087, a homolog of competence protein of Synechocystis PCC6803 and in two putative alkaline phosphatases (ALPs, Alr2234 and Alr4976), respectively. In silico analysis of All3087 is shown to possess the characteristics feature of competence proteins such as helix-hairpin-helix, N and C-terminal HKD endonuclease domain, calcium binding domain and N-terminal lipoprotein signal peptide. The SP recognition-cleavage site in All3087 was predicted (AIA-AC) using SignalP while further in-depth analysis using Pred-Lipo and WebLogo analysis for consensus sequence showed it as IAA-C. Activities of putative ALPs were confirmed by heterologous overexpression, activity assessment and zymogram analysis. ALP activity in Anabaena remains cell bound in log-phase, but during late log/stationary phase, an enhanced ALP activity was detected in extracellular milieu. The enhancement of ALP activity during stationary phase was not only due to inorganic phosphate limitation but also contributed by the presence of novel bipartite Tat-SP. The Tat signal transported the folded active ALPs to the membrane, followed by anchoring into the membrane and successive cleavage enabling transportation of the ALPs to the extracellular milieu, because of bipartite architecture and processing of transit Tat-SP.

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The wide world of non-mammalian phospholipase D enzymes
Y. Wang, M.J.O. Wakelam, V.A. Bankaitis, M.I. McDermott
Advances in Biological Regulation.2024; 91: 101000. CrossRef
In silico Analysis of Different Signal Peptides for the Excretory Production of Recombinant NS3-GP96 Fusion Protein in Escherichia coli
Shiva Mohammadi, Zohreh Mostafavi-Pour, Younes Ghasemi, Mahdi Barazesh, Soudabeh Kavousi Pour, Amir Atapour, Pooneh Mokarram, Mohammad Hossein Morowvat
International Journal of Peptide Research and Therapeutics.2019; 25(4): 1279. CrossRef
Signal Peptide Sequence Analysis of Selected Protein Sequences from Cryptosporidium parvum
Mohd Aiman Baru, Muhammad Lokman Md , Afzan Mat Yusof
Trends in Bioinformatics.2018; 11(1): 33. CrossRef

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.

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