Engineering Escherichia coli for Soluble Expression and Single Step Purification of Active Human Lysozyme.
ABSTRACT Genetically engineered variants of human lysozyme represent promising leads in the battle against drug-resistant bacterial pathogens, but early stage development and testing of novel lysozyme variants is constrained by the lack of a robust, scalable and facile expression system. While wild type human lysozyme is reportedly produced at 50-80kg per hectare of land in recombinant rice, this plant-based system is not readily scaled down to bench top production, and it is therefore not suitable for development and characterization of novel lysozyme variants. Here, we describe a novel and efficient expression system capable of producing folded, soluble and functional human lysozyme in E. coli cells. To achieve this goal, we simultaneously co-express multiple protein folding chaperones as well as harness the lysozyme inhibitory protein, Ivy. Our strategy exploits E. coli's ease of culture, short doubling time, and facile genetics to yield upwards of 30mg/L of soluble lysozyme in a bioreactor system, a 3000-fold improvement over prior efforts in E. coli. Additionally, molecular interactions between lysozyme and a his-tagged Ivy allows for one-step purification by IMAC chromatography, yielding as much as 21mg/L of purified enzyme. We anticipate that our expression and purification platform will facilitate further development of engineered lysozymes having utility in disease treatment and other practical applications.
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ABSTRACT: The cells that line the mucosa of the human gastrointestinal tract (GI, that is, oral cavity, oesophagus, stomach, small intestine, large intestine, and rectum) are constantly challenged by adverse micro-environmental factors, such as different pH, enzymes, and bacterial flora. With exception of the oral cavity, these microenvironments also contain remnant cocktails of secreted enzymes and bacteria from upper organs along the tract. The density of the GI bacteria varies, from 103/mL near the gastric outlet, to 1010/mL at the ileocecal valve, to 1011 to 1012/mL in the colon. The total microbial population (ca. 1014) exceeds the total number of cells in the tract. It is, therefore, remarkable that despite the prima facie inauspicious mixture of harmful secretions and bacteria, the normal GI mucosa retains a healthy state of cell renewal. To counteract the hostile microenvironment, the GI epithelia react by speeding cell exfoliation (the GI mucosa has a turnover time of two to three days), by increasing peristalsis, by eliminating bacteria through secretion of plasma cell-immunoglobulins and by increasing production of natural antibacterial compounds, such as defensin-5 and lysozyme. Only recently, lysozyme was found up-regulated in Barrett's oesophagitis, chronic gastritis, gluten-induced atrophic duodenitis (coeliac disease), collagenous colitis, lymphocytic colitis, and Crohn's colitis. This up-regulation is a response directed to the special types of bacteria recently detected in these diseases. The aim of lysozyme up-regulation is to protect individual mucosal segments to chronic inflammation. The molecular mechanisms connected to the crosstalk between the intraluminal bacterial flora and the production of lysozyme released by the GI mucosae, are discussed. Bacterial resistance continues to exhaust our supply of commercial antibiotics. The potential use of lysozyme to treat infectious diseases is receiving much attention.03/2014; 3(1):73-92. DOI:10.3390/pathogens3010073
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ABSTRACT: Lysozyme is a protein found in egg white, tears, saliva, and other secretions. As a marketable natural alternative to preservative, lysozyme can act as a natural antibiotic. In this study, we have isolated Bacillus lincheniformis TIB320 from soil which contains a lysozyme gene with various features. We have cloned and expressed the lysozyme in E.coli. The antimicrobial activity of lysozyme showed that it had a broad antimicrobial spectrum against several standard strains. The lysozyme can maintain efficient activities in a pH range between 3 and 9 and from 20 oC to 60 oC, respectively. The lysozyme was resistant to the pepsin and trypsin to some extent at 40 oC. Production of the lysozyme was optimized by using various expression strategies in B. subtilis WB800. The lysozyme from Baclicus lincheniformis TIB320 will be a promising choice for food additive and feed additive.Journal of Microbiology and Biotechnology 07/2014; 24(10). DOI:10.4014/jmb.1404.04039 · 1.32 Impact Factor
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ABSTRACT: A mammary gland-specific expression vector p205C3 was constructed with the 5'- and 3'-flanking regions of β-lactoglobulin gene and the first intron of β-casein gene of Chinese dairy goat as regulatory sequences. Human lysozyme (hLYZ) cDNA from mammary gland was cloned into p205C3 and the recombinant vector was used to generate transgenic mice by microinjection. Based on the lysoplate assay, four female offspring of one male founder were detected expressing recombinant hLYZ in their milk at the levels of 5-200 mg/L, and the expressed protein had the same molecular weight as that of normal hLYZ. Besides mammary glands, ectopic expressions were also found in the spleens and the small intestines of the transgenic mice. Among the offspring, the female transgenic mice maintained and expressed the transgene stably with a highest expression level of 750 mg/L. Therefore, p205C3 could be used to develop animal mammary gland bioreactors expressing hLYZ.Plasmid 09/2014; DOI:10.1016/j.plasmid.2014.09.004 · 1.76 Impact Factor