Production of Full-Length Human Pre-elafin, an Elastase Specific Inhibitor, from Yeast Requires the Absence of a Functional Yapsin 1 (Yps1p) Endoprotease

Bourbonnais, Yves; Larouche, Chantal; Tremblay, Guy M.

doi:10.1006/prep.2000.1338

Cited by 30 publications

(34 citation statements)

References 20 publications

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“…A clipped form of trappin-2 resulting from a cleavage C-terminal to Lys32 appeared together with the full-length trappin-2 after three days of fermentation. Such a proteolytic susceptibility after lysyl residues was observed by Bourbonnais et al [30] who expressed trappin-2 in Saccharomyces cerevisae. Cleavage after Lys14 and Lys36 in the so-called cementoin domain of trappin-2 was attributed unambiguously to yapsin-1, an aspartic plasma membrane protease active within the periplasmic space.…”

Section: Discussionsupporting

confidence: 56%

“…Using the culture conditions described above, we purified about 15 mgAEL )1 of each recombinant inhibitor from the yeast culture media. Using shake-flask culture conditions which give expression levels typically low relative to what is obtainable in fermenter cultures [31], we found that the amount of elafin and trappin-2 produced in our system was higher than that reported for trappin-2 expressed in similar conditions in the yeast S. cerevisiae system (2-3 mgAEL ) [30]. Though the range of expression yields is variable from one protein to another, our study confirms that the P. pastoris system allows the production of heterologous proteins at a high concentration level.…”

Section: Discussioncontrasting

confidence: 55%

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Kinetics of the inhibition of neutrophil proteinases by recombinant elafin and pre‐elafin (trappin‐2) expressed in Pichia pastoris

Zani

Nobar

Lacour

et al. 2004

European Journal of Biochemistry

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Elafin and its precursor, trappin-2 or pre-elafin, are specific endogenous inhibitors of human neutrophil elastase and proteinase 3 but not of cathepsin G. Both inhibitors belong, together with secretory leukocyte protease inhibitor, to the chelonianin family of canonical protease inhibitors of serine proteases. A cDNA coding either elafin or its precursor, trappin-2, was fused in frame with yeast a-factor cDNA and expressed in the Pichia pastoris yeast expression system. Fulllength elafin or full-length trappin-2 were secreted into the culture medium with high yield, indicating correct processing of the fusion proteins by the yeast KEX2 signal peptidase. Both recombinant inhibitors were purified to homogeneity from concentrated culture medium by one-step cationic exchange chromatography and characterized by N-terminal amino acid sequencing, Western blot and kinetic studies. Both recombinant elafin and trappin-2 were found to be fastacting inhibitors of pancreatic elastase, neutrophil elastase and proteinase 3 with k ass values of 2-4 · 10 6 M )1

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Section: Discussionsupporting

confidence: 56%

Section: Discussioncontrasting

confidence: 55%

Kinetics of the inhibition of neutrophil proteinases by recombinant elafin and pre‐elafin (trappin‐2) expressed in Pichia pastoris

Zani

Nobar

Lacour

et al. 2004

European Journal of Biochemistry

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“…The Saccharomyces cerevisiae yeast strain YBAD1 (yps1⌬::HIS3) was used for the recombinant production of pre-elafin and mutated variants. Yeast transformants were grown at 30°C in selective media as previously described (2).…”

Section: Methodsmentioning

confidence: 99%

“…The resulting plasmid, named pET32-cem, expresses an N-terminally tagged fusion protein (6 ϫ His and S-tag) composed of the bacterial thioredoxin and the cementoin domain, a cleavage site for enterokinase separating these two peptides. Yeast expression plasmids for the recombinant production of pre-elafin and the mutated variants preelafin M25K and pre-elafin M25G were described previously (2,8). Production and purification of recombinant pre-elafin and cementoin.…”

Section: Methodsmentioning

confidence: 99%

“…Production and purification of recombinant pre-elafin and cementoin. Recombinant His-tagged pre-elafin, pre-elafin M25K and pre-elafin M25G were purified to apparent homogeneity from the yeast culture supernatants, first by affinity chromatography on Ni 2ϩ /NTA resin (Qiagen Canada, Inc.), followed by cationexchange chromatography, as described previously (2). Bacterial expression of the cementoin peptide in fusion with thioredoxin (BL21 transformed with pET32-cem) was induced, from 1 liter of culture, by 1 mM IPTG (isopropyl-␤-D-thiogalactopyranoside).…”

Section: Methodsmentioning

confidence: 99%

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Human Pre-Elafin Inhibits a Pseudomonas aeruginosa -Secreted Peptidase and Prevents Its Proliferation in Complex Media

Bellemare

Vernoux

Morisset

et al. 2008

Antimicrob Agents Chemother

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Pseudomonas aeruginosa is a life-threatening opportunist human pathogen frequently associated with lung inflammatory diseases, namely, cystic fibrosis. Like other species, this gram-negative bacteria is increasingly drug resistant. During the past decade, intensive research efforts have been focused on the identification of natural innate defense molecules with broad antimicrobial activities, collectively known as antimicrobial peptides. Human pre-elafin, best characterized as a potent inhibitor of neutrophil elastase with anti-inflammatory properties, was also shown to possess antimicrobial activity against both gram-positive and gramnegative bacteria, including P. aeruginosa. Its mode of action was, however, not known. Using full-length pre-elafin, each domain separately, and mutated variants of pre-elafin with attenuated antipeptidase activity toward neutrophil elastase, we report here that both pre-elafin domains contribute, through distinct mechanisms, to its antibacterial activity against Pseudomonas aeruginosa. Most importantly, we demonstrate that the whey acidic protein (WAP) domain specifically inhibits a secreted peptidase with the characteristics of arginyl peptidase (protease IV). This is the first demonstration that a human WAP-motif protein inhibits a secreted peptidase to prevent bacterial growth in vitro. Since several WAP-motif proteins from various species demonstrate antimicrobial function with variable activities toward bacterial species, we suggest that this mechanism may be more common than initially anticipated.Pseudomonas aeruginosa is an opportunistic pathogen that is life-threatening for immunocompromised individuals and for patients suffering from chronic respiratory diseases such as cystic fibrosis (CF) (10, 27). Chronic lung P. aeruginosa infection is the primary cause of morbidity and mortality in CF patients, and its prevalence increases with age. P. aeruginosa is also the predominant bacteria associated with nosocomial infections, and acute P. aeruginosa infection may result in sepsis and death (26). P. aeruginosa is characterized by the expression of numerous virulence factors, reflected by the unusually large genome of this bacteria and has developed sophisticated mechanisms to escape the host immune defenses (10,15,27,33). In addition, due to low membrane permeability, active efflux of drugs, and the presence of ␤-lactamase activity, P. aeruginosa infections are becoming increasingly difficult to treat (17). Hence, the development of novel antimicrobial agents, preferably acting on targets exposed at the bacterial cell surface, is urgently needed.Among the P. aeruginosa virulence factors, the secreted peptidases are thought to play a critical role in tissue invasion, nutrient accessibility, and degradation of the innate host defense molecules and therefore constitute potential therapeutic targets (18)(19)(20). Because opportunistic pathogens are taking advantage of a weakened host protective shield, augmentation therapy with lung natural defense molecules also appears to be a ...

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Current Awareness

2001

Yeast

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In order to keep subscribers up‐to‐date with the latest developments in their field, this current awareness service is provided by John Wiley & Sons and contains newly‐published material on yeasts. Each bibliography is divided into 10 sections. 1 Books, Reviews & Symposia; 2 General; 3 Biochemistry; 4 Biotechnology; 5 Cell Biology; 6 Gene Expression; 7 Genetics; 8 Physiology; 9 Medical Mycology; 10 Recombinant DNA Technology. Within each section, articles are listed in alphabetical order with respect to author. If, in the preceding period, no publications are located relevant to any one of these headings, that section will be omitted. (5 weeks journals ‐ search completed 7th Feb. 2001)

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Production of Full-Length Human Pre-elafin, an Elastase Specific Inhibitor, from Yeast Requires the Absence of a Functional Yapsin 1 (Yps1p) Endoprotease

Cited by 30 publications

References 20 publications

Kinetics of the inhibition of neutrophil proteinases by recombinant elafin and pre‐elafin (trappin‐2) expressed in Pichia pastoris

Kinetics of the inhibition of neutrophil proteinases by recombinant elafin and pre‐elafin (trappin‐2) expressed in Pichia pastoris

Human Pre-Elafin Inhibits a Pseudomonas aeruginosa -Secreted Peptidase and Prevents Its Proliferation in Complex Media

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