Team:NNU-China/Proof Of Concept

Proof of concept

(Gold Medal#4)


Our team constructed the library of BL21 (DE3)-derived variant strains library by CRISPR-Cas9, and then the high throughput host screening platform was constructed. Based on this, the production 10 kinds of AMPs has been greatly improved. As proof-of-concept, we tested that whether the AMPs produced by us has antibacterial activity. Moreover, in order to extend the application scope of our project, we also applied the platform to the production of recombinant proteins. And we designed the high throughput host screening platform as the kit, which is easy and convenient to use it.

1. Purification of fusion protein and identification of antimicrobial activity

The AMP of MME and DCD-1L were chosen to purify and identify their antimicrobial activity. The fusion protein resulted from bacterium lysis was purified by Ni2+ affinity chromatography column, and then tested with 5% spacer gel and 15% separating gel for SDS-PAGE. A clear strip of about 27 kDa was obtained (Fig. 1A), which is consistent with the expected protein size. Freeze-drying is widely used in the pharmaceutical industry for the manufacture of protein drugs. About 30.4 and 42.3 mg of MME and DCD fusion protein powder were obtained per liter of culture medium, respectively. Furthermore, the thrombin was used to isolate the AMP and eGFP protein. Purified products were experimented for antimicrobial activity with agarose hole diffusion method. It was indicated from the results that purified heterozygous peptides showed significant antimicrobial activity on E. coli (Fig. 1B).

Fig. 1. A.SDS‐PAGE results of MME-37-eGFP and DCD-14-eGFP protein in BL21 (DE3).B. MME and DCD
was proved by the means of inhibiory zone to be able to kill the E. coli.
(1:H2O;2:Kan (2.5g/L); 3:AMP(4g/L); 4:MME(3.04g/L); 5: DCD(4.23g/L)).

2. Application of the high throughput host screening platform for hard-to-express protein production

Here, the hard-to-express protein of GDH was used. Using the high throughput host screening platform, five high-yielding strains were obtained, including SGDH-3, SGDH-4, SGDH-24, SGDH-42 and SGDH-46. Among them, the SGDH-42 strain exhibited a 298 folds increase than that of the wild-type strain, while SGDH-3 and SGDH-46 were 189 and 149 folds, respectively. Furthermore, in order to investigate the translation level of T7 RNAP in the high expression strains of GDH, the GFP gene was fused to the C-terminus of T7 RNAP in the genome to detect the translation level of different RBSs. Compared to the wild type, five high-expression hosts all decreased the translation level of T7 RNAP. Compared to the BL21 (DE3), the translation level of T7 RNAP in SGDH-42 was decreased by 51.5%. It was indicated that the decreased expression of T7 RNAP was contributing to the accumulation of GDH in the host. However, it is not true that increasing yields can be increased by continuously decreasing the expression intensity of T7 RNAP. For example, the SGDH-3 and SGDH-42 strains with the highest yields showed moderate translation levels. This phenomenon suggests that there was an optimal translation level of T7 RNAP, where strain growth and recombinant protein production reached a certain balance. It was verified that SGDH-42 had a significant increase in GDH yield and suppressed autolysis to some extent by SDS-PAGE. In conclusion, the library can be applied to enhance the expression of hard-to-express protein.

Fig. 2. A. The expression level of GDH and corresponding translation level of T7RNAP in five dominant strains.
B.SDS‐PAGE results of intracellular and extracellular protein in BL21 (DE3) and SGDH-42.

3. The kit of the high throughput host screening platform

Components of the kit of the high throughput host screening platform:
A: The BL21 (DE3)-derived variant strains library;
B: Ni-NTA Sefinose Resigen;
C: Thrombin-Agarose;
D: Cleavage Buffer;
E: Column.

Basic Procedure:
I Chemical Transferring
The target recombinant protein was fused with the eGFP by thrombin site and was transferred into the BL21 (DE3)-derived variant strains library.

Ⅱ Screening
The transformants is selected from plates and cultivated in 96-well plates. After, the unit cell fluorescence values of 96-well plates is tested, and strains with high fluorescence intensity value is selected and test next.

Ⅲ Verification
The selected strains are inoculated into a 30 mL TB medium containing Kan resistance and fermented in a 250 mL shaker for further verification.

Ⅳ Expand fermentation
The validated strains were inoculated into a 5L bioreactor to ferment 20h for increasing the yield of the recombinant protein.

Ⅴ Purification
The fusion protein resulted from bacterium lysis were purified by Ni2+ affinity chromatography column.

Ⅵ Enzymatic cut
The target AMPs or recombinant protein is isolated from eGFP by thrombin.

Ⅶ Product
Fig. 3. The component of kit and its basic procedure