RECOMBINANT HUMAN INSULIN-LIKE GROWTH-FACTOR BINDING PROTEIN-4, PROTEIN-5, AND PROTEIN-6 - BIOLOGICAL AND PHYSIOCHEMICAL CHARACTERIZATION

We have recently cloned cDNAs encoding human insulin-like growth facto r binding proteins (IGFBP)-4, -5 and -6 and have now expressed these B Ps in yeast as ubiquitin (Ub)-IGFBP fusion proteins. Western ligand bl otting With I-125-IGF II under nonreducing conditions of recombinant h uman (rh) IGFBP-containing yeast lysates revealed specific binding ban ds for IGFBP-4, -5, and -6 at apparent molecular masses of 24-26, 30-3 2, and 24-26 kDa, respectively, indicating expression and processing o f the fusion proteins. HPLC purified rhIGFBPs had virtually the same a mino acid composition, amino acid number, and NH2-terminal sequences a s the native BPs. Rabbit antiserum directed against each rhIGFBP-4, -5 and -6 reacted specifically with the respective rhIGFBP as well as wi th the native human counterpart and displayed very low cross-reactivit y with other IGFBPs. Except for the affinity of rhIGFBP-6 for IGF I (K (a)=8.5 x 10(8) M-1), the affinity constants of the three IGFBPs for I GF I and II lie between 1.7 and 3.3 x 10(10) M-1. When present in exce ss, rhIGFBP-4, -5, and -6 inhibited IGF I- and II-stimulated DNA and g lycogen synthesis in human osteoblastic cells, although rh-IGFBP-6 had only a weak inhibitory effect on IGF I in agreement with its relative ly lower IGF I affinity constant.