Human epidermal growth factor (hEGF) is a monomeric polypeptide consisting of 53 amino acids. The protein acts as an active mitogenic factor and affects cell growth

proliferation and differentiation by specifically binding to its cell surface receptor EGFR. The dysregulation of the hEGF gene is associated with the growth and development of certain cancers. Although hEGF protein shows a quick repair function for epidermal tissue damage and ulceration disease

some ambiguities such as short half-life and EGFR down-regulation still remain. In this study

the human EGF and porcine EGF factor were modified via directed evlotion in vitro by the staggered extension process (StEP)

in which a mutant E40V with high affinity against EGFR receptor was obtained using phage display library construction and ELISA screening. Then we used MTT

circular dichroism (CD) and wound healing assay to analyze the A431 and NIH3T3 cell proliferation

migration ability

temperature and pH tolerance of the mutant protein respectively. We found that the affinity of E40V for EGFR was significantly higher than that of wild-type EGF and the mutant could also promote the proliferation of normal cells NIH3T3

while the temperature tolerance and pH tolerance were similar to those of wildtype. Our DNA shuffling EGF with targeting properties and long halflife may provide a new application foreground for regulations of the normal cell growth and cancer targeting therapy.