Molecular cloning, overexpression, purification, and sequence analysis of the giant panda (Ailuropoda melanoleuca) ferritin light polypeptide

• Published in: Genetics and molecular research Vol. 15; no. 3
• Main Authors: Fu, L, Hou, Y L, Ding, X, Du, Y J, Zhu, H Q, Zhang, N, Hou, W R
• Format: Journal Article
• Language: English
• Published: Brazil 30-08-2016
• Subjects: Ailuropoda melanoleuca; Amino Acid Sequence; Animals; Base Sequence; Cavia porcellus; Cloning, Molecular; Conserved Sequence; Equus caballus; Escherichia coli; Felis catus; Ferritins - biosynthesis; Ferritins - genetics; Ferritins - isolation & purification; Gene Expression; Glycosylation; Isoelectric Point; Molecular Weight; Protein Processing, Post-Translational; Sequence Analysis, DNA; Sequence Analysis, Protein; Ursidae - genetics
• ISSN: 1676-5680; 1676-5680
• DOI: 10.4238/gmr.15038593

The complementary DNA (cDNA) of the giant panda (Ailuropoda melanoleuca) ferritin light polypeptide (FTL) gene was successfully cloned using reverse transcription-polymerase chain reaction technology. We constructed a recombinant expression vector containing FTL cDNA and overexpressed it in Escherichia coli using pET28a plasmids. The expressed protein was then purified by nickel chelate affinity chromatography. The cloned cDNA fragment was 580 bp long and contained an open reading frame of 525 bp. The deduced protein sequence was composed of 175 amino acids and had an estimated molecular weight of 19.90 kDa, with an isoelectric point of 5.53. Topology prediction revealed one N-glycosylation site, two casein kinase II phosphorylation sites, one N-myristoylation site, two protein kinase C phosphorylation sites, and one cell attachment sequence. Alignment indicated that the nucleotide and deduced amino acid sequences are highly conserved across several mammals, including Homo sapiens, Cavia porcellus, Equus caballus, and Felis catus, among others. The FTL gene was readily expressed in E. coli, which gave rise to the accumulation of a polypeptide of the expected size (25.50 kDa, including an N-terminal polyhistidine tag).