Molecular cloning of giant panda pituitary prolactin cDNA and its expression in Escherichia coli.
cDNA encoding pituitary (PRL) of giant panda was obtained using RT-PCR and expressed in E. coli. The results revealed that panda PRL cDNA encodes a precursor protein of 229 amino acids including a putative signal peptide of 30 amino acids and a mature protein of 199 residues with one potential N-glycosylation site. Sequence comparison indicated that panda PRL shares a high degree of identity to other known PRL sequences ranging from 98% with mink PRL to about 50% with rodent PRL. Six cysteine residues and 29 conserved residues distributed in four domains (PD1, PD2, PD3, and PD4) of PRL were observed. through multiple sequence alignment. Fourteen key residues of binding sites 1 and 2 involved in receptor binding are conserved in panda PRL. GST fused recombinant panda PRL protein was efficiently expressed with the form of insoluble inclusion bodies in E. coli BL21 transformed with a pGEX-4T-1 expression vector containing the DNA sequence encoding mature panda PRL. Western blot analysis indicated that GST-panda PRL recombinant protein could be recognized by antibody against human PRL. Our results would contribute to further elucidating the structural and functional characteristics of pituitary PRL and provide a basis for the production of recombinant panda prolactin for future use in the breeding of giant panda.
Zhang
Zhi-He
ZH
Institute of Genetics, College of Life Sciences, Zhejiang University, Hangzhou, China.
Zheng
Xu
X
Hu
Xi-lian
XL
Zhu
Mu-Yuan
MY
Hou
Rong
R
Shen
Fu-Jun
FJ
Zhang
Liang
L
Liao
Ming-Juan
MJ
Lv
Xiao-Ping
XP
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western / Q000662:veterinary; D003247:Conservation of Natural Resources; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D008969:Molecular Sequence Data; D010802:Phylogeny; D010902:Pituitary Gland / Q000378:metabolism*; D011388:Prolactin / Q000096:biosynthesis / Q000235:genetics*; D012313:RNA / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D001503:Ursidae / Q000235:genetics*
D011993:Recombinant Fusion Proteins; D012313:RNA; D011388:Prolactin