Preparation method of glutamine dipeptide
A glutamate dipeptide and alanine technology, which is applied in biochemical equipment and methods, peptides, enzymes, etc., can solve the problems of complex synthesis process, unfavorable industrial scale-up, high production cost, and achieve wide source of raw materials, easy large-scale industrial production, low cost effect
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Example Embodiment
[0023] Example 1 Construction of Amino Acid Ligase Strain
[0024] ①According to the nucleotide sequence of the amino acid ligase encoding gene ywfE of B.subtilis ATCC 15245 on Genbank, use codon software commonly used in E. coli to optimize the codon, and add the restriction site BamH I to the optimized sequence And Hind III (Sequence Listing 400 The sequence shown) was sent to Jinweizhi Company for synthesis.
[0025] ②Use Takara restriction endonucleases BamH I and Hind III to cut the target gene fragment and pET-His vector plasmid in step ① to obtain ywfE and pET-His linear fragments with the same sticky ends.
[0026] ③Use Takara T4 DNA ligase to ligate the two gene fragments in step ② to obtain the recombinant expression vector pET-His-ywfE.
[0027] ④ Transform the recombinant expression vector in step ③ into E. coli BL21 (ACCC11171) to obtain the amino acid ligase-producing strain E. coli pET-His-ywfE.
Example Embodiment
[0028] Example 2 Construction of Acetate Kinase Strain
[0029] ①Using PCR technology with the genome of Clostridium acetobutylicum ATCC 4259 as a template, according to the nucleotide sequence of the acetate kinase encoding gene ack (sequence list 400 (Shown sequence) design a pair of gene amplification primers (upstream primer is 400 The sequence shown, the downstream primer is Sequence Listing 400 Shown sequence), amplify to obtain ack gene fragment. The pair of primers respectively contain restriction sites BamH I and EcoRI.
[0030] ②Using Takara restriction enzymes BamH I and EcoRI double digestion step ① to obtain the target fragment and pET-His vector plasmid, and obtain ack and pET-His linear fragments with the same sticky ends.
[0031] ③Use Takara T4 DNA ligase to ligate the two two gene fragments obtained in step ② to obtain the recombinant expression vector pET-His-ack.
[0032] ④ Transform the recombinant expression vector of step ③ into E. coli BL21 (ACCC11171) to ob...
Example Embodiment
[0033] Example 3 Preparation of amino acid ligase and acetate kinase
[0034] ①Inoculate the strains in (1) and (2) from the glycerin preservation tube at an inoculum amount of 0.1% (v / v) to 100 mL of LB liquid medium containing ampicillin (100 μg / mL), and culture at 37°C at 200 rpm 12h, then transfer 400mL LB liquid medium containing ampicillin (100μg / mL) according to the 1% (v / v) inoculum, and continue the culture at 37°C and 200rpm. Concentration of strains OD 600nm IPTG with a final concentration of 0.1 mmol / L was added when it reached 0.6, and the protein was induced and cultured at 28°C for 10 hours.
[0035] ②After the cultivation, centrifuge the fermentation broth at 4°C and 6000rpm to collect the bacteria. After washing three times with pH 7.4 PBS buffer, resuspend in pH 7.4 PBS buffer, sonicate for 20 min, centrifuge at 4°C, 10000 rpm to obtain the supernatant.
[0036] ③Pass the supernatant through Ni at a flow rate of 1 mL / min + -After NTA resin, eluted with 50 mM imida...
PUM
Abstract
Description
Claims
Application Information
- 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.
© 2023 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap