Nisin M: a Bioengineered Nisin A Variant That Retains Full Induction Capacity but Has Significantly Reduced Antimicrobial Activity

ABSTRACT Nisin A is a potent antimicrobial with potential as an alternative to traditional antibiotics, and a number of genetically modified variants have been created that target clinically relevant pathogens. In addition to antimicrobial activity, nisin autoregulates its own production via a signal transduction pathway, a property that has been exploited in a protein expression system termed the nisin-controlled gene expression (NICE) system. Although NICE has become one of the most popular protein expression systems, one drawback is that the inducer peptide, nisin A, also has inhibitory activity. It has already been demonstrated that the N-terminal region of nisin A contributes to antimicrobial activity and signal transduction properties; therefore, we conducted bioengineering of nisin at positions Pro9 and Gly10 within ring B to produce a bank of variants that could potentially be used as alternative induction peptides. One variant, designated nisin M, has threonines at positions 9 and 10 and retains induction capacity comparable to that of wild-type nisin A, while most of the antimicrobial activity is abolished. Further analysis confirmed that nisin M produces a mix of peptides as a result of different degrees of dehydration of the two threonines. We show that nisin M exhibits potential as a more suitable alternative to nisin A for the expression of proteins that may be difficult to express or for production of proteins in strains that are sensitive to wild-type nisin. Moreover, it may address the increasing demand by industry for optimization of peptide fermentations to increase yields or production rates. IMPORTANCE This study describes the generation of a nisin variant with superior characteristics for use in the NICE protein expression system. The variant, termed nisin M, retains an induction capacity comparable to that of wild-type nisin A but exhibits significantly reduced antimicrobial activity and can therefore be used at concentrations that are normally toxic to the expression host.

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Mating in Saccharomyces cerevisiae: The Role of the Pheromone Signal Transduction Pathway in the Chemotropic Response to Pheromone

Genetics ◽

10.1093/genetics/147.1.19 ◽

1997 ◽

Vol 147 (1) ◽

pp. 19-32 ◽

Cited By ~ 5

Author(s):

Kathrin Schrick ◽

Barbara Garvik ◽

Leland H Hartwell

Keyword(s):

Signal Transduction ◽

Map Kinase ◽

Transcriptional Activation ◽

Signal Transduction Pathway ◽

Transduction Pathway ◽

Wild Type ◽

Mating Partner ◽

Map Kinase Cascade ◽

Kinase Cascade ◽

Wild Type Control

Abstract The mating process in yeast has two distinct aspects. One is the induction and activation of proteins required for cell fusion in response to a pheromone signal; the other is chemotropism, i.e., detection of a pheromone gradient and construction of a fusion site available to the signaling cell. To determine whether components of the signal transduction pathway necessary for transcriptional activation also play a role in chemotropism, we examined strains with null mutations in components of the signal transduction pathway for diploid formation, prezygote formation and the chemotropic process of mating partner discrimination when transcription was induced downstream of the mutation. Cells mutant for components of the mitogen-activated protein (MAP) kinase cascade (ste5, ste20, ste11, ste7 or fus3 kss1) formed diploids at a frequency 1% that of the wild-type control, but formed prezygotes as efficiently as the wild-type control and showed good mating partner discrimination, suggesting that the MAP kinase cascade is not essential for chemotropism. In contrast, cells mutant for the receptor (ste2) or the β or γ subunit (ste4 and stel8) of the G protein were extremely defective in both diploid and prezygote formation and discriminated poorly between signaling and nonsignaling mating partners, implying that these components are important for chemotropism.

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Mutational Activation of a Gαi Causes Uncontrolled Proliferation of Aerial Hyphae and Increased Sensitivity to Heat and Oxidative Stress in Neurospora crassa

Genetics ◽

10.1093/genetics/151.1.107 ◽

1999 ◽

Vol 151 (1) ◽

pp. 107-117

Author(s):

Qi Yang ◽

Katherine A Borkovich

Keyword(s):

Signal Transduction ◽

Neurospora Crassa ◽

Signal Transduction Pathway ◽

Sexual Cycle ◽

Intracellular Camp ◽

Wild Type ◽

Independent Functions ◽

Aerial Hyphae ◽

Mutational Activation ◽

Camp Levels

Abstract Heterotrimeric G proteins, consisting of α, β, and γ subunits, transduce environmental signals through coupling to plasma membrane-localized receptors. We previously reported that the filamentous fungus Neurospora crassa possesses a Gα protein, GNA-1, that is a member of the Gαi superfamily. Deletion of gna-1 leads to defects in apical extension, differentiation of asexual spores, sensitivity to hyperosmotic media, and female fertility. In addition, Δgna-1 strains have lower intracellular cAMP levels under conditions that promote morphological abnormalities. To further define the function of GNA-1 in signal transduction in N. crassa, we examined properties of strains with mutationally activated gna-1 alleles (R178C or Q204L) as the only source of GNA-1 protein. These mutations are predicted to inhibit the GTPase activity of GNA-1 and lead to constitutive signaling. In the sexual cycle, gna-1R178C and gna-1Q204L strains are female-fertile, but produce fewer and larger perithecia than wild type. During asexual development, gna-1R178C and gna-1Q204L strains elaborate abundant, long aerial hyphae, produce less conidia, and possess lower levels of carotenoid pigments in comparison to wild-type controls. Furthermore, gna-1R178C and gna-1Q204L strains are more sensitive to heat shock and exposure to hydrogen peroxide than wild-type strains, while Δgna-1 mutants are more resistant. In contrast to Δgna-1 mutants, gna-1R178C and gna-1Q204L strains have higher steady-state levels of cAMP than wild type. The results suggest that GNA-1 possesses several Gβγ-independent functions in N. crassa. We propose that GNA-1 mediates signal transduction pathway(s) that regulate aerial hyphae development and sensitivity to heat and oxidative stresses, possibly through modulation of cAMP levels.

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Production and Purification of Secretory Simian Cytidine Monophosphate-N-acetylneuraminic Acid Hydroxylase Using Baculovirus-Protein Expression System

Biological and Pharmaceutical Bulletin ◽

10.1248/bpb.b15-00299 ◽

2015 ◽

Vol 38 (8) ◽

pp. 1220-1226 ◽

Cited By ~ 1

Author(s):

Tadanobu Takahashi ◽

Sawako Kawagishi ◽

Hiroki Funahashi ◽

Nonoka Hayashi ◽

Takashi Suzuki

Keyword(s):

Protein Expression ◽

Expression System ◽

Acetylneuraminic Acid ◽

Protein Expression System ◽

Cytidine Monophosphate

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Optimization of a Fusion Protein Expression System using Human Cell Lines to Create a Practical Immunoassay Reagent

KAGAKU KOGAKU RONBUNSHU ◽

10.1252/kakoronbunshu.41.38 ◽

2015 ◽

Vol 41 (1) ◽

pp. 38-42 ◽

Cited By ~ 2

Author(s):

Kounosuke Hayashi ◽

Yusuke Tomozoe ◽

Kenji Nagai ◽

Kyoichi Matsuba ◽

Masayuki Mitsumori ◽

...

Keyword(s):

Fusion Protein ◽

Protein Expression ◽

Cell Lines ◽

Human Cell ◽

Expression System ◽

Human Cell Lines ◽

Fusion Protein Expression ◽

Protein Expression System

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Construction and characterization of mutated LEA peptides in Escherichia coli to develop an efficient protein expression system

Journal of Molecular Recognition ◽

10.1002/jmr.2658 ◽

2017 ◽

Vol 31 (1) ◽

pp. e2658 ◽

Cited By ~ 2

Author(s):

Nishit Pathak ◽

Hiro Hamada ◽

Shinya Ikeno

Keyword(s):

Escherichia Coli ◽

Protein Expression ◽

Expression System ◽

Protein Expression System

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Expression of Recombinant Fusion Protein of Newcastle Disease Virus from Escherichia coli Plasmid Clone C-2a by In-vitro Cell-free Protein Expression System

BIO Web of Conferences ◽

10.1051/bioconf/20202004004 ◽

2020 ◽

Vol 20 ◽

pp. 04004

Author(s):

Ahmad Pandu Satria Wiratama ◽

Aris Haryanto

Keyword(s):

Protein Expression ◽

Newcastle Disease ◽

Recombinant Plasmid ◽

Expression System ◽

Free Protein ◽

F Protein ◽

E Coli ◽

Protein Expression System ◽

Sds Page

Newcastle Disease Virus (NDV) is an infectious disease that infect many kinds of wild and domesticated birds. Infection of NDV become a massive problem for poultry industry around the world especially in Indonesia. Vaccination is an effort to prevent the infection of NDV in poultry. NDV vaccine that used in Indonesia is a conventional life vaccine from LaSota and B1 strains. These type of vaccine is 21%-23% genetically distinct with the virus that spread in the environment. The antibody protection provided by the vaccine is not effective. Therefore, vaccination with new local NDV strain is needed to prevent the NDV infection in Indonesia. The previously study research reported that the local isolate of NDV from Kulon Progo, Indonesia has been isolated. Fusion (F) protein encoding gene that has been inserted into pBT7-N-His expression p lasmid which isolated from clone C-2a of E. coli, then it was expressed by the Cell-free protein expression system. The aim of this study was to confirm whether clone C-2a of E.coli carrying a recombinant plasmid pBT7-N-His-Fusion NDV and to express a recombinant F protein of NDV in-vitro from expression plasmid by cell-free protein expression system. This work started by detection of recombinant plasmid pBT7-N-His-Fusion NDV by DNA plasmid extraction followed by agarose gel electrophoresis. The recombinant F protein was in-vitro expressed by cell-free protein expression kit. The expressed F protein of NDV then was visualized by SDS-PAGE and Westernblott to analyse the expression of NDV recombinant F protein. It confirmed that clone C-2a of E. coli contained plasmid pBT7-N-His (4.001 bp) inserted by recombinant F protein of NDV gene (642 bp). The visualisation of expressed recombinant F protein by SDS-PAGE and Westernblott showed the NDV recombinant F protein was a specific protein fragment with molecular weight of 25,6 kDa..

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