Isolated polypeptide and application thereof
A peptide segment and precursor peptide technology, applied in the field of bioengineering, can solve the problems of low yield and inability to meet production needs, and achieve the effect of increasing yield
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0058]In this example, construct the plasmids of several key components that regulate the production of nisin, the specific method is as follows
[0059] (1) Genomic DNA from Lactococcus lactis was obtained using Blood & Cell Culture DNA Mini Kit (QIAGEN, Hileden, Germany) according to the instructions.
[0060] (2) Amplify the nisB (SEQ ID No: 6), nisC (SEQ ID No: 7) and nisP (SEQ ID No: 8) genes from the genome by PCR, and synthesize the nisZ sequence nisZ (SEQ ID No: 5) by gene , and then subcloning nisZ into pJL1 through suitable restriction sites, and cloning nisB, nisC, and nisP into pET28a to construct expression plasmids for nisZ, nisB, nisC, and nisP. The primers used and related plasmid information are shown in Table 1, respectively. and Table 2. Utilize the primers in Table 1 and Table 2 to amplify the corresponding genes from the ATCC11454 yeast genome by PCR, and use the corresponding enzyme cutting sites in Table 2 to digest the PCR amplified fragments, and simu...
Embodiment 2
[0070] Using the nisZ, nisB, nisC and nisP expression plasmids prepared in Example 1 to rebuild the nisin synthesis pathway in vitro, the specific method is as follows:
[0071] 1. Preparation of the main components of the reconstructed nisin in vitro reaction system (CFPS)
[0072] (1) NisB and NisC proteins:
[0073] (a) E. coli BL21(DE3) cells were freshly transformed with plasmids pYL02 and pYL03, and single-colony transformants were grown overnight at 37°C in 50 mL medium supplemented with 50 μg / mL kanamycin.
[0074] (b) Transfer 2L LB medium according to 1% inoculum size until OD600 reaches 0.6-0.8, then cool the culture to 18°C, add IPTG to a final concentration of 0.5mM (NisB) or 0.2mM (NisC) to induce, wherein , for NisC overexpression, add an additional 100 μM ZnCl 2 to ensure its activity.
[0075] (c) After the culture from step (b) continued to grow in culture for 20 hours, the cells were harvested by centrifugation at 5000 g for 20 minutes at 4°C and resuspen...
Embodiment 3
[0098] Carry out quantitative detection to nisin, the method is as follows:
[0099] (1) Determination of nisin activity by the agar diffusion method, specifically as follows, by adding 50 mg of commercially available nisin 106 I U / g (Sigma-Aldrich) to 50 mL of sterile 0.02 mol / L HCl to prepare a stock nisin solution (2000 IU / mL).
[0100] (2) Standard nisin solutions of 1000, 500, 250, 200, 100, 20 and 5 IU / mL were prepared using the nisin stock solution, diluted with 0.02 mol / L HCl and used to construct a standard curve.
[0101] (3) Preparation of bioassay medium containing 1.2% tryptone, 0.75% yeast extract, 0.75% NaCl, 0.3% NaH 2 PO 4 and 2% agar, after adding sterile 0.75% glucose and 0.5% Tween 20 to the bioassay medium, the agar medium was obtained, the agar medium was cooled to 50°C, and inoculated with 1.5% indicator strain Micrococcus luteus The overnight culture of NCIB 8166 was plated, several wells were drilled on each plate after sufficient solidification, a...
PUM
Login to view more Abstract
Description
Claims
Application Information
Login to view more - R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap