Expression and bioactivity analysis of TNF30, a TNFα nanobody, in Escherichia coli

Figures & data

Figure 1. Inductive expression and identification of the recombinant His₆-tagged TNF30 in E. coli. (A) SDS-PAGE detection of the products. (B) Western bolt analysis of the products using anti-His₆ antibodies.

Figure 2. Optimisation of the culture medium for the soluble expression of the recombinant His₆-tagged TNF30. (A) SDS-PAGE analysis of the products fermented in different media. (B) Relative quantification of the recombinant His₆-tagged TNF30 fermented in different media.

Figure 3. Purification of the recombinant His₆-tagged TNF30. Lane M: protein maker (Protein Pre-stained Marker, Thermos); Lane1: supernatant of the recombinant cell lysate; Lane 2: flow-through; Lanes 3–8: gradient elution with imidazole at concentration of 20, 40, 70, 130, 250 and 500 mmol/L, respectively; Lane9: cell lysate supernatant of E. coli Rosetta (DE3) harbouring a pET28a empty plasmid.

Figure 4. Affinity assay of recombinant TNF30 with TNFα. (A) Products of strains transformed with the empty vector. (B) Recombinant products of strains harbouring His₆-fused TNF30.

Table 1. Anti-inflammation activity of TNF30 in the carrageenan-induced mice paw oedema model (X±SD; n = 8).

Time (h)	Control	TNF30 (30 mg/kg)	TNF30 (60 mg/kg)	Aspirin
0	0	0	0	0
1	13.76 ± 1.19	12.53 ± 1.09	12.91 ± 1.71	10.08 ± 1.17**
2	16.47 ± 2.97	13.84 ± 1.16*	11.03 ± 1.34**	9.97 ± 0.76**
3	16.65 ± 1.22	13.28 ± 1.58**	10.77 ± 0.59**	10.84 ± 1.21**
4	17.24 ± 2.83	10.11 ± 2.41**	10.51 ± 1.82**	10.05 ± 1.08**
5	10.75 ± 0.98	9.83 ± 0.85	4.95 ± 1.13**	5.06 ± 1.37**
6	7.91 ± 1.02	7.23 ± 1.31	2.06 ± 1.64**	1.38 ± 1.25**

Notes: *P<0.05, **P<0.01 vs. the inflammatory mice treated with PBS.

Figure 5. Anti-inflammatory activity of TNF30 in vivo in a carrageenan-induced mice paw oedema experiment. Histopathological analysis of normal mice (A) and inflammatory mice treated with PBS (B), TNF30 at 30 mg/kg (C) or TNF30 at 60 mg/kg (D).