Method for efficiently expressing lysostaphin

A technology encoded by lysostaphin and staphylococcus enzyme, which is applied in the field of genetic engineering, can solve the problems such as the difficulty of predicting the processing and folding process of foreign proteins

Pending Publication Date: 2020-10-09
GUANGDONG VTR BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The Pichia pastoris expression system is one of the most commonly used protein expression systems, but it is difficult to predict the post-translational processing and folding process of foreign proteins expressed in the host, so whether a specific foreign protein can be efficiently expressed in the Pichia pastoris expression system remains to be determined. Overcome unexpected technical difficulties

Method used

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  • Method for efficiently expressing lysostaphin
  • Method for efficiently expressing lysostaphin
  • Method for efficiently expressing lysostaphin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] The construction of embodiment 1 lysostaphin expression bacterial strain

[0019] The lysostaphase amino acid sequence of Staphylococcus simulans (simulating Staphylococcus) was obtained from NCBI database, as shown in SEQ ID NO.1. Mature region of lysostaphin (amino acid sequence As shown in SEQ ID NO.2, the gene sequence is codon-optimized (as shown in SEQ ID NO.11), and a stop codon TAA is added at the end of the gene sequence to obtain a mature lysostaphin gene sequence, codon-optimized The gene sequence is shown in SEQ ID NO.3, SEQ ID NO.12, and SEQ ID NO.13.

[0020] Lysostaphin amino acid, SEQ ID NO.1

[0021]

[0022] Mature region of lysostaphin, SEQ ID NO.2

[0023]

[0024] Codon-optimized gene sequence: SEQ ID NO.3

[0025]

[0026]

[0027] SEQ ID NO.12

[0028]

[0029] SEQ ID NO.13

[0030]

[0031]

[0032] Taking the optimized gene sequence SEQ ID NO.3 as an example to introduce the vector construction, other gene sequences ca...

Embodiment 2

[0049] Expression and detection of embodiment 2 recombinant lysostaphin

[0050] Shake flask culture: Pick the Pichia lysostaphin expression strain constructed in Example 1, inoculate it in 5mL of LYPD medium, and culture it at 30°C and 220rpm for 24 hours to prepare a seed solution. Inoculate 50mL BMGY culture medium (250mL Erlenmeyer flask) according to 1% inoculum amount, cultivate at 30°C, 220rpm for 24 hours, centrifuge at 4000Xg for 5min, collect the bacteria, resuspend the bacteria in 50mL BMMY medium, cultivate at 30°C, 220rpm, every Add 1% methanol (final concentration) for 24 hours to induce, induce for 3 days, centrifuge at 4000Xg for 5min, and take the supernatant for detection.

[0051] Fermentation in a fermenter: Pick the Pichia lysostaphin expressing strain constructed in Example 1, inoculate it in 25 mL of YPD medium, and culture it at 30° C. at 220 rpm for 24 hours to prepare a primary seed solution. Inoculate 20 mL of primary seed liquid into 200 mL of BMGY...

Embodiment 3

[0056] Embodiment 3 stability evaluation

[0057] Strain stability was evaluated by serial subculture. The Pichia cell colony expressing lysostaphin constructed in Example 1 was picked and inoculated in 3 mL of YPD medium, and cultured at 30° C. and 220 rpm for 24 hours to prepare a seed solution. Take 0.5mL seed solution and inoculate in 25mL BMGY medium, and culture at 30°C and 220rpm for 24 hours. Transfer the bacterial solution to a 50mL centrifuge tube, centrifuge (4000×g, room temperature) to discard the supernatant, resuspend the bacterial cells with 25mL BMMY medium with a methanol concentration of 1%, transfer the bacterial solution to a 250mL Erlenmeyer flask, 30°C, 220rpm Incubate for 72 hours, adding methanol to a concentration of 1% every 24 hours. Centrifuge (4000×g, room temperature) to take the fermentation supernatant for detection of inhibition zone and protein concentration. The selected strains were selected and subcultured for 10 generations, and the in...

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Abstract

The invention relates to the field of gene engineering, in particular to a method for efficiently expressing lysostaphin. According to the invention, secretory expression of lysostaphin is carried outby using a pichia pastoris expression system, massive expression of lysostaphin is realized, thus reducing production costs; the highest expression quantity of lysostaphin reaches 6g/L; the lysostaphin is secreted and expressed in the fermentation supernatant, cells do not need to be broken, thalli only need to be removed through simple filtration, and lysostaphin crude enzyme liquid with few impure proteins can be obtained; the lysostaphin sample prepared through the method has an obvious inhibiting effect on staphylococcus, especially staphylococcus aureus.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for highly expressing lysostaphin. Background technique [0002] A staph infection is an infection caused by the staphylococcus bacteria. Lysosthaphin (Lysosthaphin, EC3.4.24.75) is an endopeptidase that can cut off the five-glycine peptide bridge structure in the peptidoglycan of the cell wall, thereby rapidly lysing bacteria. Because the cell wall of Staphylococcus is rich in pentaglycine peptide bridge structure, lysostaphin has a particularly significant bactericidal effect on Staphylococcus, especially on drug-resistant Staphylococcus aureus MRSA, and also has a certain antibacterial effect on other Gram-positive bacteria. Lysostaphin hydrolyzes the bacterial cell wall so that the bacteria cannot grow normally, thereby achieving a bactericidal effect. This unique antibacterial mechanism determines that it is not easy to produce drug resistance. [0003] Lysostap...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/52C12N15/81C12N15/57C12R1/84
CPCC12N9/52C12N15/815C12N2800/22C12Y304/24075
Inventor 梁伟凡李阳源唐雪梅容晓燕邓智远刘金山邓敬阳林影
Owner GUANGDONG VTR BIO TECH
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