• Animal cells and systems
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• v.16 no.6
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• pp.455-461
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• 2012

Amongst the various antimicrobial peptides, lysozyme plays a central role in initiating and maintaining the antibacterial defense response of insect. Here we propose the biosynthesis and refolding of recombinant lysozyme in Escherichia coli expressed in inclusion body form. The Agrius lysozyme gene was amplified using gene specific primers and then ligated into the pGEX-4T-1 vector, which contained the glutathione S-transferase (GST) gene as a fusion partner. A recombinant lysozyme was expressed in E. coli Rosetta cells using a pGEX-4T-1 expression vector, and the fusion protein was induced by ioporpyl-${\beta}$-D-thiogalactopyranoside (IPTG). The recombinant protein produced as an inclusion body was resolubilized in solubilization buffer, and the resultant solution was dialyzed in refolding buffer. After thrombin cleavage, the recombinant lysozyme was purified by ion exchange chromatography and reverse phase chromatography. The recombinant lysozyme was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and immunoreactivity against the anti-Agrius lysozyme was observed by western blot analysis of this protein. The recombinant lysozyme displayed antibacterial activity against Bacillus megaterium and Micrococcus luteus, which was confirmed by the inhibition zone assay.

• Animal cells and systems
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• v.12 no.3
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• pp.149-155
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• 2008

We have isolated and characterized Agrius convolvuli cDNA encoding a c-type lysozyme. The cDNA sequence encodes a processed protein of 139 amino acid residues with 19 amino acid residues amino-terminal signal sequence and 120 amino acid residues mature sequence. The amino acid residues responsible for the catalytic activity and the binding of the substrate are conserved. Agrius lysozyme has a high identity to Manduca sexta. Recombinant A. convolvuli lysozyme was expressed in Escherichia coli BL21(DE3) pLysS cells for pGEX 4T-1 expression vector. Their optimal conditions for the fusion protein expression and purification were screened. Lysozyme gene amplified with primers ACLyz BamHI and ACLyz XhoI was ligated into the pGEX 4T-1 vector, which contained the glutathione S-transferase(GST) gene for fusion partner. The fusion protein was induced by IPTG and identified by SDS-PAGE analysis. Molecular weight of the fusion protein was estimated to be about 45 kDa. Recombinant lysozyme, fused to GST, was purified by glutathion-Sepharose 4B affinity chromatography. Western blot analysis of this protein revealed an immunoreactivity with the anti-Agrius lysozyme.

• Korean Journal of Veterinary Service
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• v.44 no.4
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• pp.247-256
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• 2021

The recombinant lysozyme-HJL34 proteins were expressed and purified using commercial Escherichia (E.) coli expression system. Stx2e⁺ F18⁺ E. coli, Actinobacillus pleuropneumoniae (APP), Streptococcus (S.) suis, and Clostridium (C.) perfringens strains were isolated from pigs. The minimum inhibitory concentrations (MICs) of the recombinant lysozyme-HJP34 proteins were examined by means of the microtiter plate method, according to the NCCLS recommendations. The possibility of its as the alternatives to antibiotics was tested in piglets. The MICs were determined as 75 ㎍/mL, 300 ㎍/mL, 75 ㎍/mL, 35.5 ㎍/m against Stx2e⁺ F18⁺ E. coli, APP, S. suis, C. perfringens, respectively. A total of 25 piglets were divied 5 groups. The piglets in group A~C were fed with commercial feed and those in groups D, E were fed with commercial feedstuff. All piglets in groups B~E were challenged with virulent Stx2e⁺ F18⁺ E. coli, APP, S. suis strains. Groups C and D were treated with antimicrobial from 24 h after challenge. All piglets in group B died within 3 days after challenge. Among 5 piglets in groups C and D piglets, 80% survived after challenge. Among group E piglets, 60% were alive until the end of this study. Therefore, this study indicates that recombinant lysozyme-HJP34 proteins is a suitable possibility as a feed additive for reduction of diseases by bacterial pathogens in piglet feed.

• Biotechnology and Bioprocess Engineering:BBE
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• v.7 no.1
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• pp.52-55
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• 2002

Flow cytometric techniques were used to investigate cell size, protein content and cell cycle behavior of recombinant Saccharomyces cerevisiae strains producing human lysozyme (HLZ). Two different signal sequences, the native yeast $MF\alpha1$ signal sequence and the rat $\alpha-amylase$ signal sequence, were used for secretion of HLZ. The strain containing the rat $\alpha-amylase$ signal sequence showed a higher level of internal lysozyme and lower specific growth rates. Flow cytometric analysis of the total protein content and cell size showed the strain harboring the native yeast signal sequence had a higher total protein content than the strain containing the rat $\alpha-amylase$ signal sequence. Cell cycle analysis indicated that the two lysozyme producing recombinant strains had an increased number of cells in the $G_2+M$ phase of the yeast cell cycle compared with the host strain SEY2102.

• Asian-Australasian Journal of Animal Sciences
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• v.27 no.4
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• pp.574-579
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• 2014

A lysozyme gene from breast of Tibetan sheep was successfully expressed by secretion using a-factor signal sequence in the methylotrophic yeast, Pichia pastoris GS115. An expression yield and specific activity greater than 500 mg/L and 4,000 U/mg was obtained. Results at optimal pH and temperature showed recombinant lysozyme has higher lytic activity at pH 6.5 and $45^{\circ}C$. This study demonstrates the successful expression of recombinant lysozyme using the eukaryotic host organism P. pastoris paving the way for protein engineering. Additionally, this study shows the feasibility of subsequent industrial manufacture of the enzyme with this expression system together with a high purity scheme for easy high-yield purification.

• Molecules and Cells
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• v.42 no.3
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• pp.262-269
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• 2019

The porcine myeloid antimicrobial peptide (PMAP), one of the cathelicidin family members, contains small cationic peptides with amphipathic properties. We used a putative lysozyme originated from the bacteriophage P22 (P22 lysozyme) as a fusion partner, which was connected to the N-terminus of the PMAP36 peptide, to markedly increase the expression levels of recombinant PMAP36. The PMAP36-P22 lysozyme fusion protein with high solubility was produced in Escherichia coli. The final purified yield was approximately 1.8 mg/L. The purified PMAP36-P22 lysozyme fusion protein exhibited antimicrobial activity against both Gram-negative and Grampositive bacteria (Staphylococcus aureus, Salmonella enterica serovar Typhimurium, Pseudomonas aeruginosa, and Bacillus subtilis). Furthermore, we estimated its hemolytic activity against pig erythrocytes as 6% at the high concentration ($128{\mu}M$) of the PMAP36-P22 lysozyme fusion protein. Compared with the PMAP36 peptide (12%), our fusion protein exhibited half of the hemolytic activity. Overall, our recombinant PMAP36-P22 lysozyme fusion protein sustained the antimicrobial activity with the lower hemolytic activity associated with the synthetic PMAP36 peptide. This study suggests that the PMAP36-P22 lysozyme fusion system could be a crucial addition to the plethora of novel antimicrobials.

• Asian-Australasian Journal of Animal Sciences
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• v.28 no.12
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• pp.1774-1783
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• 2015

Milk lysozyme is the ubiquitous enzyme in milk of mammals. In this study, the cDNA sequence of a new chicken-type (c-type) milk lysozyme gene (YML), was cloned from yak mammary gland tissue. A 444 bp open reading frames, which encodes 148 amino acids (16.54 kDa) with a signal peptide of 18 amino acids, was sequenced. Further analysis indicated that the nucleic acid and amino acid sequences identities between yak and cow milk lysozyme were 89.04% and 80.41%, respectively. Recombinant yak milk lysozyme (rYML) was produced by Escherichia coli BL21 and Pichia pastoris X33. The highest lysozyme activity was detected for heterologous protein rYML5 (M = 1,864.24 U/mg, SD = 25.75) which was expressed in P. pastoris with expression vector $pPICZ{\alpha}A$ and it clearly inhibited growth of Staphylococcus aureus. Result of the YML gene expression using quantitative polymerase chain reaction showed that the YML gene was up-regulated to maximum at 30 day postpartum, that is, comparatively high YML can be found in initial milk production. The phylogenetic tree indicated that the amino acid sequence was similar to cow kidney lysozyme, which implied that the YML may have diverged from a different ancestor gene such as cow mammary glands. In our study, we suggest that YML be a new c-type lysozyme expressed in yak mammary glands that plays a role as host immunity.

• Journal of Microbiology and Biotechnology
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• v.9 no.5
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• pp.576-581
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• 1999

Effects of signal sequences, protein sizes and dissolved oxygen on the secretion of human lysozyme from a recombinant yeast were experimentally characterized. The systems consisted of Saccharomyces cerevisiae host SEY2102 that was transformed with two different plasmids. These plasmids were identical with an exception to the plasmid pMC614, which contained the native yeast MFα1 sequence and the plasmid pMC632 with the non-native rat α-amylase signal sequence. The expression of human lysozyme was controlled by the ADHI promoter. The native yeast MFαl signal sequence was more efficient than the non-native rat α-amylase signal sequence in directing the secretion of human lysozyme. Lysozyme secreted with the α-amylase signal was retained inside the cells and released to the medium very slowly, thereby causing a lower cell growth rate and a decreased product secretion rate. Lysozyme was secreted more efficiently than invertase, which is an order of magnitude bigger in molecular size compared to lysozyme, which was under the direction of the MFαl signal sequence, suggesting that protein sizes may affect the secretion efficiency. When expressed in anaerobic conditions in the medium where the ADHI promoter was derepressed, the amount of lysozyme secreted was about twice higher than that of the aerobic culture. However, the secretion rates were identical. This result showed that the dissolved oxygen level may affect the efficiency of protein secretion only, and not the secretion rate of the product protein.

• Journal of the Korean Society of Food Science and Nutrition
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• v.28 no.2
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• pp.348-352
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• 1999

We have intended to accelerate the secretion of human lysozyme(HLY) with permeabilizing agents from the cultivated cells of the recombinant Saccharomyces cerevisiae. The five agents CaCl2, Tween 80, ethanol, Triton X 100, and cetyltrimethylammonium bromide(CTAB) were used as permeabilizing agents. Treatments of the yeast cell with CaCl2, Tween 80, and ethanol were effective to increase the secretion from the yeast cells. Especially, treatment of 10% ethanol increased the extracellular HLY activity by 38.6% at 30oC for 48 h in culture broth. But Triton X 100 and CTAB unexpectedly didn't play a role in increase of HLY secretion. Recovery of a foreign protein by permeabilizing agents is easier than by osmotic shock, and is less expensive than enzymatic digestion.

• Animal cells and systems
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• v.15 no.1
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• pp.29-36
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• 2011

Insect lysozymes are basic, cationic proteins synthesized in fat body and hemocytes in response to bacterial infections and depolymerize the bacterial cell wall. The c-type lysozyme of the insect Spodoptera litura (SLLyz) is a single polypeptide chain of 121 residues with four disulfide bridges and 17 rare codons and is approximately 15 kDa. The full-length SLLyz cDNA is 1039 bp long with a poly(A) tail, and contains an open reading frame of 426 bp long (including the termination codon), flanked by a 54 bp long 5' UTR and a 559 bp long 3' UTR. As a host for the production of high-level recombinant proteins, E. coli is used most commonly because of its low cost and short generation time. However, the soluble expression of heterologous proteins in E. coli is not trivial, especially for disulfide-bonded proteins. In order to prevent inclusion body formation, GST was selected as a fusion partner to enhance the solubility of recombinant protein, and fused to the amplified products encoding mature SLLyz. The expression vector pGEX-4T-1/rSLLyz was then transformed into E. coli BL21(DE3)pLysS for soluble expression of rSLLyz, and the soluble fusion protein was purified successfully. Inhibition zone assay demonstrated that rSLLyz showed antibacterial activity against B. megaterium. These results demonstrate that the GST fusion expression system in E. coli described in this study is efficient and inexpensive in producing a disulfide-bonded rSLLyz in soluble, active form, and suggest that the insect lysozyme is an interesting system for future structural and functional studies.