Cassette structure of FGF constructs and strategy of mutagenesis. (A) Full-length FGF-2 and partial-length FGF-1 cDNA fragments were initially synthesized in 4 cassettes (A, B, C, and D) and ligated as indicated. The amino acid changes (Glu → Lys [E → K], His → Tyr [H → Y], Ala → Thr [A → T]) are shown above the FGF-2 sequence and were inserted to generate specific cleavage sites for the restriction enzymes indicated in parentheses. Numbering of residues is based on that used in the published crystal structures of FGF-1 and FGF-2.^26-30  (B) Construction of FGF chimeric mutants. The mutants were subcloned by using indicated restriction enzymes. (C) Construction of FGF-2 deletion mutants. Deletion mutants were generated by PCR and cloned into pET26bFGF-2 with appropriated restriction enzyme as indicated, and the resulting constructs were ABC, BCD, ABD, and ACD. Gray shading in B and C represents FGF-2 fragments; white sections, FGF-1 fragments.