Molecular cloning, functional expression, and mutagenesis of cDNA encoding a cysteine proteinase inhibitor from sunflower seeds

Sunflower cystatin Scb differs from other phytocystatins in that it is a highly basic protein with a pI value of 9.6 and includes six additional amino acids (Arg³⁰-Leu-Gln-Arg-Thr³⁴, Thr³⁷) in the middle region as compared with other phytocystatins. We identified and sequenced a complete cDNA encoding the Scb; the cDNA of Scb consists of 645 nucleotides and includes an open reading frame encoding a polypeptide of 123 amino acids. On the basis of these findings, Scb appears to be synthesized as a prepeptide consisting of a signal sequence of 22 amino acids and a mature protein of 101 amino acids. A recombinant Scb (rScb) was produced by expression in Escherichia coli and purified by gel filtration on Sephacryl S-200 followed by ion-exchange column chromatography on a S-Sepharose column. rScb exhibited almost the same inhibitory activity toward papain as the authentic Scb did, but its inhibition profile toward cathepsins B, L, and H was slightly different. Scb mutant proteins, in which selected N-terminal residues or the additional amino acids were deleted, were subsequently constructed and characterized with respect to their inhibitory activities toward papain. The result revealed that the additional sequence (Arg³⁰-Leu-Gln-Arg-Thr³⁴) in Scb is not essential for papain-inhibitory activity, while the N-terminal amino acids (Ile¹-Pro²) as well as the N-terminal glycine residues Gly³ and/or Gly⁴ play an important role in manifesting the inhibitory activity toward papain.