Purification, Cloning, and Identification of Two Thaumatin-like Protein Isoforms in Jelly Fig (Ficus awkeotsang) Achenes

• Published in: Journal of agricultural and food chemistry 2007-09, Vol.55 (18), p.7602-7608
• Main Authors: Chua, Anna C. N, Chou, Wing-Ming, Chyan, Chia-Lin, Tzen, Jason T. C
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Biological and medical sciences; Cloning, Molecular; complementary DNA; Ficus; Ficus - chemistry; Ficus awkeotsang; Food additives; Food industries; Fruit and vegetable industries; fruits (food); Fundamental and applied biological sciences. Psychology; Gene Expression; General aspects; jelly figs; molecular cloning; Molecular Sequence Data; pathogenesis-related proteins; plant proteins; Plant Proteins - chemistry; Plant Proteins - genetics; Plant Proteins - isolation & purification; polypeptides; Protein Isoforms - chemistry; Protein Isoforms - genetics; Protein Isoforms - isolation & purification; purification; Seeds - chemistry; Sequence Alignment; thaumatin-like proteins
• ISSN: 0021-8561; 1520-5118
• DOI: 10.1021/jf071553x

Jelly curd used for a popular summer drink in Taiwan is prepared by extracting the pericarpial portion of jelly fig (Ficus awkeotsang Makino) achenes. The two most abundant proteins found in jelly curd have been identified as a pectin methylesterase and a chitinase. A method was developed to purify the next abundant protein by 40% ammonium sulfate precipitation and flowing through Mono Q chromatography. In sodium dodecyl sulfate−polyacrylamide gel electrophoresis analyses, the purified protein migrated as a polypeptide of 20 kDa in the absence of β-mercaptoethanol but split into a minor polypeptide of 20 kDa and a major polypeptide of 27 kDa in the presence of this reducing agent. Two cDNA fragments encoding precursor polypeptides of two putative thaumatin-like protein isoforms were obtained by polymerase chain reaction cloning and subsequently overexpressed in Escherichia coli to generate recombinant proteins for antibody preparations. Immunological detection and mass spectrometric analyses indicated that the two split polypeptides were thaumatin-like protein isoforms encoded by the two cloned cDNA fragments. Keywords: Achene; jelly fig (Ficus awkeotsang); mass spectrometric analyses; pericarp; thaumatin-like protein