Expression purification of endolysin

["Collaborating Expert | Jie Huang, M.S.", "Biology University of Science and Technology of China"], ["Reviewed by | Dr. Nie Yifeng", "Nanobiomedicine University of Chinese Academy of Sciences"]

Summary

As a phage-derived enzyme, endolysin is essentially a protein molecule that assists in the release of phage particles from the bacterial host by breaking down the peptidoglycan component of the bacterial cell wall, and can be expressed during phage replication, disrupting key chemical bonds in the peptidoglycan of the bacterial cell wall.

Principle

Through the protein expression and purification of endolysin, it is possible to clone the endolysin gene into an expression vector (e.g., pET28a), which is further transformed into E. coli for amplification, and the recombinant plasmid is induced to be expressed by IPTG, obtaining endolysin proteins purified by a nickel column.

Appliance

Potential solutions for the treatment of antibiotic-resistant bacterial infections.

Operation method

purification of endolysin expression

Principle

Materials and Instruments

Instruments:

Centrifuge, Ultrasonic Crusher

Constant temperature shaker, constant temperature incubator, PCR instrument, electrophoresis apparatus

Gel scanner, nickel column purification system, 0.22 μm filter membrane

Reagents:

Plasmid Extraction Kit, Gel Recovery Kit

Nucleic Acid Endonuclease, T4 Ligase, Pfu Polymerase

IPTG, Kaomas Brilliant Blue

Move

(1) Cloning of endolysin

1. Primer design: complete the genome sequencing and annotation of the endolysin to be studied, design primers P1 and P2 according to the measured genome, and introduce the enzyme cleavage sites.

2. PCR amplification of endolysin gene: the reaction system and program are shown in Table 1.

Table 1 Reaction system

3. PCR reaction conditions: pre-denaturation at 95 ℃ for 5 min, denaturation at 95 ℃ for 30 s, annealing at Tm for 30 s, extension at 72 ℃ for 30 s for 30 cycles, final extension at 72 ℃ for 10 min, and re-reaction at 26 ℃ for 2 min (termination).

4. Recovery of PCR product gel: Agarose gel electrophoresis was performed, and 50 μL of the product was sampled. The target gene strip was cut off, and the target gene was recovered using a gel recovery kit (commonly available in laboratories).

5. Plasmid extraction: use the plasmid extraction kit to extract the vector plasmid (common brands are available in general laboratories).

6、Enzymatic cleavage of the site: use the enzyme cleavage site designed at the beginning of the vector and the target gene for double enzyme cleavage, and react at 37 ℃ for 3 h.

7、Cutting gel recovery: The digested product was subjected to agarose gel electrophoresis, and the target bands containing DNA fragments were cut out and recovered, and the concentration of DNA was detected and stored in the refrigerator at -20 ℃ for spare.

8、Linking of DNA and vector: Use T4 ligase to link vector and DNA fragments, and overnight at 16 ℃.

9. Transformation of E. coli: Take 50 μL of E. coli BL21 receptor cells, add 5 μL of ligand solution and incubate on ice for 30 min, shake well, then heat shock at 42 ℃ for 90 s, and then rapidly ice bath for 2 min, add 1 mL of pre-warmed LB medium after the ice bath, and then resuscitate at 37 ℃ for 1 h, and then finally apply the plate to kanamycin-resistant plate, and then incubate at 37 ℃ for the night. The plates were incubated overnight at 37 ℃. On the next day, single colonies were picked for colony PCR, and the positive colonies were inoculated into the medium and incubated overnight at 37 ℃.

10. Extract the plasmid and carry out double enzyme digestion for verification (see steps 5 and 6).

(2) Endolysin expression and purification

1, a small amount of induced expression: select a single colony overnight culture, overnight bacteria inoculated into 1 mL of medium, 37 ℃ incubated for 2~3 h, then add 10 μL of 0.1 M IPTG at 16 ℃ induced for 4 h, 20 μL of bacterial liquid added 7.5 μL of protein buffer sampling solution, 100 ℃ boiled water for 10 min, electrophoresis, poured into the Thomas Brilliant Blue overnight shaking, cleaned to look for the presence of target protein bands, further expansion of the culture. After washing, search for the presence of target protein bands and further expand the culture.

2、Expanded culture: spread the positive colonies on the plate overnight, select single colonies and inoculate them into 10 mL of medium, incubate at 37 ℃ for 6 h, inoculate them into 500 mL of culture medium according to the ratio of 1:100, shake the bed at 37 ℃ until the OD600 value is 0.6~0.8, add IPTG inducer with final concentration of 1 mM, and continue to incubate for 12 h at 16 ℃.

3, cell fragmentation: the bacterial liquid was centrifuged for 10 min (4000 g, 4 ℃), resuspended in 30 mL of PBS, and ultrasonically fragmented by adding PMSF and tritonX-100 in an ice bath for 1 h. The fragmented bacterial liquid was transferred to a 50 mL centrifuge tube at 4 ℃, centrifuged at 12000 rpm for 10 min, and the supernatant was filtered for impurities with a 0.22 μm filter membrane.

4. Protein purification: Ni-NTA purification system was used to purify the target protein by nickel column affinity chromatography, and the purity of the protein was analyzed by electrophoresis after purification.

Caveat

1. PCR conditions and procedures need to be mapped out on a gradient according to the experiment. 2.

2. T4 ligase is unstable at high temperatures, usually ligated overnight at 16 ℃, and can be maintained at room temperature around 25 ℃ for seven to eight hours.

Common Problems

Poor protein-induced expression (protein-induced expression conditions do not necessarily apply to all labs, you need to do your own experiments to figure out the conditions).

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