Epitope-tagged insulin-like growth factor-I expression in muscle

Development of a recombinant insulin like growth factor I (ICF-I) that is d istinguishable from its endogenous counterpart would provide a powerful too l for delineating the role of IGF in myogenesis. Therefore, the objective o f this study was to create an epitope-tagged IGF-I that retains biological activity and determine whether expression of this construct is possible in muscle tissue following direct DNA injection. Expression vectors were creat ed that encoded porcine IGF-I containing a T7 (11-amino acid) epitope-tag ( TIGF). Immunoreactivity of the purified recombinant TIGF was confirmed usin g monoclonal antibodies. Biological activity was evaluated by examining dif ferentiation of myoblasts cultured with TIGF or transfected with TIGF plasm id DNA. Addition of purified TIGF to myoblast cultures stimulated (P < 0.05 ) muscle creatine kinase levels similar to insulin (10(-5) M). Likewise, tr ansfection of L6A1 with TIGF DNA hastened (P < 0.01) differentiation compar ed to control pcDNA-transfected myoblasts. The integrity of the recombinant protein was confirmed using a sandwich-configured enzyme linked immunosorb ent assay. Finally, recombinant TIGF DNA was injected in porcine muscle and the ability to detect TIGF protein was evaluated. TIGF expression was dete cted in muscle fibers of injected porcine muscle. These data show that a T7 amino acid tag placed on the amino terminus of the TGF-I protein remains i ntact during processing and does not interfere with the biological activity of the molecule. Use of this DNA construct is an excellent tool for invest igating the role of IGFs in control muscle development and provides a model to investigate other regulators of animal growth. (C) 2000 Elsevier Scienc e Inc. All rights reserved.