Secretion of active human lysozyme by acremonium chrysogenum using a fusarium alkaline protease promoter system

Morita, Shigeru; Kuriyama, Masato; Nakatsu, Masanori; Suzuki, Masaru; Kitano, Kazuaki

doi:10.1016/0168-1656(95)00051-q

Cited by 14 publications

(5 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Hence, a secretory expression system in Saccharomyces cerevisiae using chicken lysozyme secretion signal sequence was devised for successful production of soluble and fully active enzyme [16,17]. After the functional production in S. cerevisiae, other successful high-level expression systems of recombinant human lysozyme using various different hosts, including Aspergillus oryzae [18], Acremonium chrysogenum [19], Pichia pastoris [20,21], rice [22], transgenic mice [23,24] and transgenic cattle [25], followed. The highest level in the milk of transgenic mice reached 1.76 g/l, which is three times higher than human whey [24].…”

Section: Development Of Efficient Recombinant Production Systems Using Several Types Of Expression Hostsmentioning

confidence: 99%

Untitled

2014

Integr Mol Med

View full text Add to dashboard Cite

A variety of proteins with potential for medical applications contain disulfide-bridges. In this review, the author describes investigations of such proteins, particularly those concerned with clarification of structure -function relationships and molecular engineering, focusing on three classes of targets, namely human lysozyme, plant lectins composed of hevein-type domains and extracellular domains of human Fas ligand and Fas receptor. With each class of proteins, biological functions relevant to therapeutic applications, structural features, development of sample preparation methods and the experimental results for the clarification of structure -function relationships, including those of protein engineering studies on the basis of their three-dimensional structures, is summarized in the light of our current knowledge. The studies were performed by elucidating details in structural and functional consequences after introducing ligand complex formations, site-directed mutagenesis and site-specific chemical modifications, which employed various biochemical and biophysical analyses in combination with newly developed recombinant expression and chemical synthesis methods. The main findings from the studies include basic principles concerning the structure -function relationships in the enzymatic catalysis by endoglycosidases and carbohydrate recognition by lectins as well as biotechnological advances in the use of extracellular domains of death ligands and death receptors. The experimental results reviewed here will contribute to the future development of novel disulfide-bridged proteins with therapeutic usefulness targeting unmet medical needs.

show abstract

Section: Development Of Efficient Recombinant Production Systems Using Several Types Of Expression Hostsmentioning

confidence: 99%

Untitled

2014

Integr Mol Med

View full text Add to dashboard Cite

show abstract

“…5 Today, lysozyme is mainly used in biochemical research and has multiple clinical applications, such as the treatment of herpes and warts. [6][7][8][9] So far, some methods, such as ion exchange 10 and resonance light scattering, 11,12 have been used for the determination of lysozyme. However, these methods suffer from poor selectivity, low sensitivity, poor anti-interference ability and expensive equipment.…”

Section: Introductionmentioning

confidence: 99%

A chemiluminescence sensor for determination of lysozyme using magnetic graphene oxide multi-walled carbon nanotube surface molecularly imprinted polymers

Wang

Duan

et al. 2016

RSC Adv.

View full text Add to dashboard Cite

show abstract

“…Therefore, the hen egg-white lysozyme is the most readily available form of commercial lysozyme, since it is relatively inexpensive compared to human lysozyme (3). However, the hLY is the body's own protein and has a natural compatibility; therefore, it may exhibit greater safety than other lysozymes in clinical trials (7). Furthermore, the hLY has two and a half times higher antimicrobial activity than the hen egg-white lysozyme (8).…”

Section: Introductionmentioning

confidence: 99%

Cloning and functional expression of a human lysozyme gene (hly) from human leukocytes in Pichia pastoris

Wei

Tang

et al. 2012

Mol Med Report

View full text Add to dashboard Cite

Abstract. hly is a cDNA gene derived from human leukocytes that encodes a mature human lysozyme (abbreviated to hLY). The aim of the present study was to determine the effect of cloned hly on recombinant hLY (r-hLY) activity under optimized conditions. hly was amplified by RT-PCR and ligated into the pPIC9K plasmid. The cloned cDNA (hly) was 393 bp in length, encoding a 130 amino acid hLY with a calculated molecular mass of 14,698 Da. The recombinant expression plasmid, designated as pPIC9K-hly, was linearized with SacⅠ and transformed into Pichia pastoris GS115 (his4, Mut + ) by electroporation. The integration of hly into the P. pastoris genome was confirmed by PCR analysis using 5'-AOX1 and 3'-AOX1 primers. Yeast extract peptone dextrose (YPD) plates containing different concentrations of geneticin (G418) were used for the screening of P. pastoris transformants (His + , Mut + ) with multiple hly copies. One transformant resistant to 4.0 mg/ ml of G418, designated as P. pastoris GShLY4-6, expressing the highest r-hLY activity was selected by the shake-flask test, and used for the optimization of expression conditions. When the P. pastoris GShLY4-6 was induced under optimized conditions, the expressed r-hLY activity was up to 533 U/ml, which was 1.52 times as high as that (351 U/ml) expressed using the standard protocol. SDS-PAGE assay demonstrated that the r-hLY with an apparent molecular mass of approximately 14.7 kDa was extracellularly expressed in P. pastoris. In conclusion, r-hLY increased following the cloning of hly and the optimized conditions as compared to standard protocol. IntroductionHuman lysozyme (EC 3.2.1.17) is a bacteriolytic enzyme that is widely distributed in a variety of tissues and body fluids (1). It hydrolyzes preferentially the β-1,4 glycosidic linkages between the N-acetylmuramic acid and N-acetylglucosamine residues that occur in the peptidoglycan cell wall structures of certain microorganisms, particularly those of Gram-positive bacteria, and therefore appears to have a role in host defense (2). The hLY comprises 130 amino acid residues and belongs to the group of lysozymes called c-type lysozymes (3). Due to its bacteriolytic activity, it is considered to be useful for medical and food uses. There is a reemergence of uses of lysozyme in various clinical trials. The addition of lysozyme to baby formulas was proposed (4), and lysozyme had also been shown to inhibit the growth of HIV-1 in vitro. Of note, the anti-HIV activity of lysozyme is independent of its muramidase activity (5). In addition, findings of previous studies have shown that it is effective in inactivating spores of Bacillus cereus in cheese by causing high hydrostatic pressure following the addition of lysozyme (6).Currently, hLY is mainly extracted from human milk and placenta, which is restricted by many factors, such as the lack of raw materials and high cost of purification. Therefore, the hen egg-white lysozyme is the most readily available form of commercial lysozyme, since it is relatively inexpensive compared...

show abstract

Secretion of active human lysozyme by acremonium chrysogenum using a fusarium alkaline protease promoter system

Cited by 14 publications

References 25 publications

Untitled

Untitled

A chemiluminescence sensor for determination of lysozyme using magnetic graphene oxide multi-walled carbon nanotube surface molecularly imprinted polymers

Cloning and functional expression of a human lysozyme gene (hly) from human leukocytes in Pichia pastoris

Contact Info

Product

Resources

About