Secretion of biologically active human epidermal growth factor from Escherichia coli using Yersinia pestis Caf1 signal peptide.
OBJECTIVE
The Caf1 secretion pathway of Yersinia pestis is one of the most well-characterized export machineries. To facilitate the secretion of human epidermal growth factor (hEGF) in Escherichia coli, a DNA fragment containing the synthetic gene for hEGF was joined to a sequence encoding the signal peptide of Yersinia pestis Caf1 protein.


METHODS
The gene for hEGF was synthesized by overlapping polymerase chain reaction technique and was placed under the control of the caf1 gene promoter in the recombinant plasmid pHL401 which was used to transfect E. coli BL-21 for production of hEGF. The biological function of recombinant hEGF was measured by estimating its ability to stimulate the proliferation of human embryonic kidney-293 cells.


RESULTS
The results indicated that the expressed hybrid protein was processed during the secretion process. The majority of the mature hEGF was recovered from the periplasm and medium fractions, with a small amount of the expressed hEGF deposited in the cytoplasm. Furthermore, it was found that the cell proliferation was enhanced by the recombinant hEGF.


CONCLUSIONS
These results suggested that the recombinant hEGF was successfully secreted through the inner membrane of cells into the periplasm and then through the outer membrane into the medium via the action of the signal peptide of Y. pestis Caf1 in E. coli. The mitogenic activity of hEGF in cells was demonstrated.
Liu
Yi-Lin
YL
Department of Dermatology, Taipei Veterans General Hospital, Taipei, Taiwan.
Huang
Liang-Min
LM
Lin
Wen-Po
WP
Tsai
Chung-Chin
CC
Lin
Tsung-Shun
TS
Hu
Yu-Hua
YH
Chen
Hquan-Shin
HS
Han
Jun-Ming
JM
Wang
Hsian-Jenn
HJ
Liu
Yu-Tien
YT
D001426:Bacterial Proteins / Q000096:biosynthesis*; D002473:Cell Wall / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000096:biosynthesis*; D004926:Escherichia coli / Q000378:metabolism*; D006801:Humans; D018832:Molecular Chaperones / Q000096:biosynthesis*; D019897:Periplasm / Q000378:metabolism; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis
D001426:Bacterial Proteins; D018832:Molecular Chaperones; D011993:Recombinant Fusion Proteins; C070195:CAf1M protein, Yersinia pestis; D004815:Epidermal Growth Factor