cDNA cloning of growth hormone from giant panda (Ailuropoda melanoleuca) and its expression in Escherichia coli.
A cDNA encoding Ailuropoda melanoleuca growth hormone (AmGH) was isolated from pituitary total RNA using RT-PCR and expressed in Escherichia coli. This is the first report of a GH nucleotide and amino acid (aa) sequence from giant panda. The open reading frame of AmGH (651 bp) encodes a precursor of 216 aa comprising a 26 aa signal peptide and a 190 aa mature protein with four cysteine residues similar to the typical primary structure of mammalian GH precursor. AmGH shares a high degree of identity (54-98.9%) with that of mammals, birds and amphibians, but a very low identity with bony fish GH (only 20-30%). The mature AmGH exhibits striking similarity to that of putative ancestral GH with a difference of only two residues, indicating a very slow basal rate of molecular evolution. The DNA fragment encoding mature AmGH was then subcloned into the pGEX-4T-1 expression vector and highly expressed in E. coli host BL21 with IPTG induction. The expressed proteins fused to GST were found to be sequestered into inclusion bodies and therefore the NaOH method was employed to solubilize the inclusion bodies; the proteins were further purified by Glutathione Sepharose 4B affinity chromatography. The production and purification of GST-AmGH reported here provide a basis for further studies on the biological activity of AmGH.
Liao
Ming Juan
MJ
State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou, 310012 PR China.
Zhu
Mu Yuan
MY
Zheng
Xu
X
Zhang
Zhi He
ZH
Zhang
An Ju
AJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D013006:Growth Hormone / Q000096:biosynthesis / Q000235:genetics*; D007544:Isopropyl Thiogalactoside; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D001503:Ursidae / Q000235:genetics*
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D007544:Isopropyl Thiogalactoside; D013006:Growth Hormone