Expression and purification of human epidermal growth factor (hEGF) fused with GB1

Figures & data

Table 1. Synthesized coding sequences and primers used for the construction of GB1–hEGF fusion plasmid.

Name	Sequence (5’→3’)
Coding sequence of His6–GB1–TEV	CACCATCATCATCATCACCAGTACAAGCTTGCTCTGAACGGTAAAACCCTGAAAGGTGAAACCACCACCGAAGCTGTTGACGCTGCTAC CGCGGAAAAAGTTTTCAAACAGTACGCTAACGACAACGGTGTTGACGGTGAATGGACCTACGACGACGCTACCAAAACCTTCACGGTA ACCGAAGAAAATCTGTATTTCCAG
Coding sequence of hEGF	AATAGTGACTCTGAATGTCCCCTGTCCCACGATGGGTACTGCCTCCATGATGGTGTGTGCATGTATATTGAAGCATTGGACAAGTATGCA TGCAACTGTGTTGTTGGCTACATCGGGGAGCGATGTCAGTACCGAGACCTGAAGTGGTGGGAACTGCGC
Primer: His6-GB1-TEV-F (Nde I)	CGGGTTAACCATATGCACCATCATCATCATCACCA
Primer: His6-GB1-TEV-R (BamH I)	GTTGGATCCCTGGAAATACAGATTTTCTT
Primer: hEGF-F (BamH I)	GTTGGATCCAATAGTGACTCTGAATGT
Primer: hEGF-R (Xho I)	CCGCTCGAGCTAGCGCAGTTCCCACCACTT

Figure 1. Engineered fusion sequence containing GB1, N-terminal hexahistidine (His₆) tag and C-terminal TEV protease recognition site.

Figure 2. Construction of the pHGB1–TEV plasmid.

Figure 3. Recombinant hEGF with different fusion proteins: Schematics (A); arrows indicate different enzymes cleavage sites. SDS-PAGE analysis (B) of expression level and solubility of hEGF with different fusion proteins; arrows indicate each recombinant hEGF.

Figure 4. Tricine–SDS-PAGE analysis of purified hEGF.

Figure 5. Mitogenic activity analysis of different concentrations of hEGF to the NIH-3T3 cells in vitro.