Leader Peptide Establishes Dehydration Order, Promotes Efficiency, and Ensures Fidelity During Lacticin 481 Biosynthesis

Key functional elements of the vector (promoter, leader and terminator regions) that provide the expression of a target l,3-l,4-(3-glucanase gene from Rhizomucor miehei in the Komagataella kurtzmanii yeast have been optimized. It was shown that the promoter regions of the gene AOX1 from the Pichia pastoris yeast currently reclassified as Komagataella phaffti and from К. kurtzmanii yeast as parts of a vector provided equal levels of expression of the target gene in the cells of the recipient strain К. kurtzmanii Y727his4, i.e. they were completely interchangeable. This means that genetic constructs that were previously developed for the biosynthesis of recombinant proteins in К. phajfii are able to provide an effective expression in the К kurtzmanii yeast. The leader peptide MF4I (used as a variant of mif4I containing one amino acid substitution) and the leader peptide maxHH (containing the double proregion of the Hspl50 protein from Saccharomyces cerevisiae) confirmed the status of the most powerful elements among the five leader sequences analyzed. Their efficiency was 1.7 times higher than that of the standard leader from the yeast alpha-factor, and by 20% higher than the characteristics of the second group of artificial leaders. At the same time, it was found that, the choice of the terminator region had the strongest influence on the expression of the target gene among all of the vector functional elements. The best terminator elements were variants derived from the transcription termination region of the AOX1 gene, and the difference in the expression level of the target gene using different terminators was approximately 4.5 times. Based on the analysis of the obtained data, the optimal composition of the key functional elements of the expression vector was determined ; it included the promoter and terminator regions of the AOX1 yeast gene and one of the artificial leaders, mif4I or maxHH. β-glucanase, Komagataella kurtzmanii, yeast, secretion, strain producer The work was financially supported by the Ministry of Science and Higher education of the Russian Federation (Unique Project Identifier RFMEFI60717X0179) using the Unique Scientific Facility of the National Bio-Resource Center «All-Russian Collection of Industrial Microorganisms», NRC «Kurchatov Institute» - GOSNIIGENETIKA

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Much of the Microcin J25 Leader Peptide is Dispensable

Journal of the American Chemical Society ◽

10.1021/ja910191u ◽

2010 ◽

Vol 132 (8) ◽

pp. 2514-2515 ◽

Cited By ~ 44

Author(s):

Wai Ling Cheung ◽

Si Jia Pan ◽

A. James Link

Keyword(s):

Leader Peptide ◽

Microcin J25

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Induction of erm(C) Expression by Noninducing Antibiotics

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01266-07 ◽

2008 ◽

Vol 52 (3) ◽

pp. 866-874 ◽

Cited By ~ 52

Author(s):

Marne Bailey ◽

Tobin Chettiath ◽

Alexander S. Mankin

Keyword(s):

Narrow Range ◽

Colorimetric Detection ◽

State Level ◽

Leader Peptide ◽

23S Rrna ◽

Macrolide Antibiotics ◽

Reporter Expression ◽

Pronounced Increase ◽

Control Drug ◽

Diffusion Assay

ABSTRACT Ketolides, which represent the newest macrolide antibiotics, are generally perceived to be noninducers of inducible erm genes. In the study described in this paper we investigated the effects of several macrolide and ketolide compounds on the expression of the inducible erm(C) gene by Escherichia coli cells. Exposure to 14-member-ring macrolide drugs and to azithromycin led to a rapid and pronounced increase in the extent of dimethylation of Erm(C) target residue A2058 in 23S rRNA. When cells were incubated with subinhibitory concentrations of ketolides, the extent of A2058 dimethylation was also increased, albeit to a lower level and with kinetics slower than those observed with macrolides. The induction of erm(C) expression by ketolides was further confirmed by using a reporter construct which allows the colorimetric detection of induction in a disc diffusion assay. Most of the ketolides tested, including the clinically relevant compounds telithromycin and cethromycin, were able to induce the reporter expression, even though the induction occurred within a more narrow range of concentrations compared to the concentration range at which induction was achieved with the inducing macrolide antibiotics. No induction of the reporter expression was observed with 16-member-ring macrolide antibiotics or with a control drug, chloramphenicol. The deletion of three codons of the erm(C) leader peptide eliminated macrolide-dependent induction but left ketolide-dependent induction unchanged. We conclude that ketolides are generally capable of inducing erm genes. The narrow range of ketolide inducing concentrations, coupled with the slow rate of induction and the lower steady-state level of ribosome methylation, may mask this effect in MIC assays.

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Recombinant Protein Truncation Strategy for Inducing Bactericidal Antibodies to the Macrophage Infectivity Potentiator Protein of Neisseria meningitidis and Circumventing Potential Cross-Reactivity with Human FK506-Binding Proteins

Infection and Immunity ◽

10.1128/iai.01815-14 ◽

2014 ◽

Vol 83 (2) ◽

pp. 730-742 ◽

Cited By ~ 16

Author(s):

Magdalena K. Bielecka ◽

Nathalie Devos ◽

Mélanie Gilbert ◽

Miao-Chiu Hung ◽

Vincent Weynants ◽

...

Keyword(s):

Neisseria Meningitidis ◽

Bactericidal Activity ◽

Binding Proteins ◽

Leader Peptide ◽

Type I ◽

Globular Domain ◽

His Tag ◽

Macrophage Infectivity Potentiator ◽

Fk506 Binding Proteins ◽

Macrophage Infectivity

A recombinant macrophage infectivity potentiator (rMIP) protein ofNeisseria meningitidisinduces significant serum bactericidal antibody production in mice and is a candidate meningococcal vaccine antigen. However, bioinformatics analysis of MIP showed some amino acid sequence similarity to human FK506-binding proteins (FKBPs) in residues 166 to 252 located in the globular domain of the protein. To circumvent the potential concern over generating antibodies that could recognize human proteins, we immunized mice with recombinant truncated type I rMIP proteins that lacked the globular domain and the signal leader peptide (LP) signal sequence (amino acids 1 to 22) and contained the His purification tag at either the N or C terminus (C-term). The immunogenicity of truncated rMIP proteins was compared to that of full (i.e., full-length) rMIP proteins (containing the globular domain) with either an N- or C-terminal His tag and with or without the LP sequence. By comparing the functional murine antibody responses to these various constructs, we determined that C-term His truncated rMIP (−LP) delivered in liposomes induced high levels of antibodies that bound to the surface of wild-type but not Δmipmutant meningococci and showed bactericidal activity against homologous type I MIP (median titers of 128 to 256) and heterologous type II and III (median titers of 256 to 512) strains, thereby providing at least 82% serogroup B strain coverage. In contrast, in constructs lacking the LP, placement of the His tag at the N terminus appeared to abrogate bactericidal activity. The strategy used in this study would obviate any potential concerns regarding the use of MIP antigens for inclusion in bacterial vaccines.

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MPTP delta, a putative murine homolog of HPTP delta, is expressed in specialized regions of the brain and in the B-cell lineage

Molecular and Cellular Biology ◽

10.1128/mcb.13.9.5513-5523.1993 ◽

1993 ◽

Vol 13 (9) ◽

pp. 5513-5523

Author(s):

K Mizuno ◽

K Hasegawa ◽

T Katagiri ◽

M Ogimoto ◽

T Ichikawa ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

Type A ◽

Extracellular Domain ◽

Fine Tuning ◽

Leader Peptide ◽

Reticular Nucleus ◽

Cdna Clones ◽

Protein Tyrosine ◽

Type C

Protein tyrosine phosphatases (PTPs), together with protein tyrosine kinases (PTKs), are involved in the regulation of cell activation, growth, and differentiation. To further elucidate the fine tuning of cell growth and differentiation through tyrosine phosphorylation, we tried to isolate mouse receptor-type PTP (RPTP) cDNA clones by screening mouse brain cDNA libraries with mouse CD45 PTP domain probes under reduced-stringency conditions. Characterization of isolated cDNA clones for RPTP showed that the cytoplasmic region contains two tandem repeats of PTP domain of about 230 amino acids with intrinsic phosphatase activity. The extracellular region was composed of immunoglobulin (Ig)-like domains and fibronectin type III (FN-III)-like domains. The gene was highly homologous to human PTP delta (HPTP delta) and thus was named MPTP delta (murine counterpart of HPTP delta). The MPTP delta gene appeared to generate at least three species of mRNA, which differ in the composition of the extracellular domain: type A, one Ig-like and four FN-III-like domains; type B, one Ig-like and eight FN-III-like domains; and type C, three Ig-like and eight FN-III-like domains. Interestingly, the 5' untranslated region and the leader peptide of types A and B were completely different from those of type C. Northern (RNA) blot analysis demonstrated that brain, kidney, and heart cells express three mRNA species of about 7 kb. Antibody directed against part of the extracellular domain of type A MPTP delta recognized a 210-kDa protein in brain and kidney lysates. In situ hybridization of brain samples revealed that MPTP delta mRNA is present in the hippocampus, thalamic reticular nucleus, and piriform cortex, where some Src family PTKs have been also demonstrated to exist. Although MPTP delta mRNA was not detected in lymphoid tissues, all of the pre-B-cell lines tested and one of three B-cell lines tested expressed MPTP delta mRNA, whereas antibody-producing B-cell hybridomas and T-cell and macrophage lines did not. Finally, the MPTP delta locus was tightly linked to the brown (b) locus on mouse chromosome 4.

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tRNA(Trp) translation of leader peptide codon 12 and other factors that regulate expression of the tryptophanase operon.

Journal of Bacteriology ◽

10.1128/jb.172.6.3100-3107.1990 ◽

1990 ◽

Vol 172 (6) ◽

pp. 3100-3107 ◽

Cited By ~ 41

Author(s):

P Gollnick ◽

C Yanofsky

Keyword(s):

Leader Peptide

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Insights into AMS/PCAT transporters from biochemical and structural characterization of a double Glycine motif protease

eLife ◽

10.7554/elife.42305 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 19

Author(s):

Silvia C Bobeica ◽

Shi-Hui Dong ◽

Liujie Huo ◽

Nuria Mazo ◽

Martin I McLaughlin ◽

...

Keyword(s):

Structural Data ◽

Leader Peptide ◽

Gram Negative Bacteria ◽

Protease Domain ◽

Peptide Recognition ◽

Gram Positive Bacteria ◽

Dual Functional ◽

Signaling Peptides

The secretion of peptides and proteins is essential for survival and ecological adaptation of bacteria. Dual-functional ATP-binding cassette transporters export antimicrobial or quorum signaling peptides in Gram-positive bacteria. Their substrates contain a leader sequence that is excised by an N-terminal peptidase C39 domain at a double Gly motif. We characterized the protease domain (LahT150) of a transporter from a lanthipeptide biosynthetic operon in Lachnospiraceae and demonstrate that this protease can remove the leader peptide from a diverse set of peptides. The 2.0 Å resolution crystal structure of the protease domain in complex with a covalently bound leader peptide demonstrates the basis for substrate recognition across the entire class of such transporters. The structural data also provide a model for understanding the role of leader peptide recognition in the translocation cycle, and the function of degenerate, non-functional C39-like domains (CLD) in substrate recruitment in toxin exporters in Gram-negative bacteria.

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The Borrelia burgdorferi 37-Kilodalton Immunoblot Band (P37) Used in Serodiagnosis of Early Lyme Disease Is the flaA Gene Product

Journal of Clinical Microbiology ◽

10.1128/jcm.37.3.548-552.1999 ◽

1999 ◽

Vol 37 (3) ◽

pp. 548-552 ◽

Cited By ~ 15

Author(s):

Robert D. Gilmore ◽

Rendi L. Murphree ◽

Angela M. James ◽

Sarah A. Sullivan ◽

Barbara J. B. Johnson

Keyword(s):

Lyme Disease ◽

Borrelia Burgdorferi ◽

Genomic Library ◽

Disease Patient ◽

Humoral Response ◽

Immunoglobulin M ◽

Leader Peptide ◽

Serum Samples ◽

Present Generation ◽

Periplasmic Flagella

The 37-kDa protein (P37) of Borrelia burgdorferi is an antigen that elicits an early immunoglobulin M (IgM) antibody response in Lyme disease patients. The P37 gene was cloned from aB. burgdorferi genomic library by screening with antibody from a Lyme disease patient who had developed a prominent humoral response to the P37 antigen. DNA sequence analysis of this clone revealed the identity of P37 to be FlaA, an outer sheath protein of the periplasmic flagella. Recombinant P37 expression was accomplished inEscherichia coli by using a gene construct with the leader peptide deleted and fused to a 38-kDa E. coli protein. The recombinant antigen was reactive in IgM immunoblots using serum samples from patients clinically diagnosed with early Lyme disease that had been scored positive for B. burgdorferi anti-P37 reactivity. Lyme disease patient samples serologically negative for theB. burgdorferi P37 protein did not react with the recombinant. Recombinant P37 may be a useful component of a set of defined antigens for the serodiagnosis of early Lyme disease. This protein can be utilized as a marker in diagnostic immunoblots, aiding in the standardization of the present generation of IgM serologic tests.

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CONSTRUCTION, EXPRESSION AND PURIFICATION OF RECOMBINANT PRE-MATURE PEPTIDE OF PLANTARICIN F FROM Lactobacillus plantarum S34 IN Escherichia coli

Indonesian Journal of Agricultural Science ◽

10.21082/ijas.v16n1.2015.31-38 ◽

2015 ◽

Vol 16 (1) ◽

pp. 31

Author(s):

Kusdianawati Kusdianawati ◽

Apon Zaenal Mustopa ◽

Suharsono Suharsono ◽

Bugi Ratno Budiarto ◽

Fatimah Fatimah ◽

...

Keyword(s):

Escherichia Coli ◽

Lactobacillus Plantarum ◽

Proteolytic Enzymes ◽

Leader Peptide ◽

Human Consumption ◽

Mature Peptide ◽

Expression And Purification ◽

Food Preservative ◽

E Coli ◽

Sds Page

Plantaricin is one of bacteriocins that have the potential to be used as food preservative. Plantaricin is safe for human consumption because it can be easily degraded by proteolytic enzymes. The objective of this study was to express and purify recombinant pre-mature peptide of plantaricin F from Lactobacillus plantarum S34 in Escherichia coli. Plantaricin gene-specific primer was used to obtain pln F structural gene amplicon from L. plantarum S34. This amplicon was cloned in pET32a vector and expressed in E. coli BL21 (DE3) pLysS. Pre-mature plantaricin F peptide was expressed as Histagged-fusion protein and separated by Co2+-chelating affinity chromatography. L. plantarum S34-derived pre-mature plantaricin F peptide fused with thioredoxin-(His)6tag had successfully been expressed in E. coli BL21 (DE3) pLysS using pET32a as an expression vector. The fused recombinant pln F as pre-mature state expressed had a molecular mass of +24 kDa, meanwhile the fused recombinant that contained only the leader peptide of pln F appeared as +20 kDa based on SDS-PAGE separations. The optimal production of fused recombinant pln F as soluble fraction was obtained when culture condition was added with 0.5 mM of IPTG and incubated at 22°C for 5 hours (OD~1). Furthermore, the expression of fused recombinant pln F as its pre-mature peptide pointed out that the pln F’s leader peptide could be proteolytically cleaved by a system in heterologous cells. Overall, heterologous pln F production as pre-mature peptide fused with thioredoxin-(His)6tag had been well established. From this research, we expect plantaricin F can be expressed and purified in E. coli.

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