Molecular cloning, sequence characterization and heterologous expression of buffalo (Bubalus bubalis) oviduct-specific glycoprotein in E. coli.
Oviductin is a high molecular weight oviduct-specific glycoprotein secreted by the non-ciliated epithelial cells of oviduct during estrous cycle and early pregnancy. It plays an important role during fertilization and early embryonic development. The oviductin gene from oviductal tissues of buffalo was successfully cloned and sequenced. The sequence analysis revealed that buffalo and cattle oviductin share very high homology between their cDNA sequences. The predicted amino acid sequences of the buffalo oviductin exhibited the highest percent of identity of 97 % with bovine followed by 94 % with goat, 93 % with sheep, 78 % with porcine, 72 % with human, 67 % with hamster and rabbit and 65 % with mouse. Oviductin was also observed to share high similarity with the mammalian chitinase, however oviductins do not show chitinase activity due to Glu→Ile mutation in the active site responsible for chitinase activity. The phylogenetic tree based on amino acid sequences of oviductin indicated that buffalo oviductin was closely related to its cattle counterpart, and this clustering is in accordance with the classic taxonomic relationship. Tissue specific expression of the transcripts for buffalo oviductin revealed a high level expression in oviduct and ovary followed by testis, mammary gland, kidney, while in mammary epithelial cells and liver its expression was very low. The full length matured oviductin and its domains constituting chitinase-like domain and mucin-like domain were cloned into pET and pGEX series of expression vectors and over expressed in E. coli. The soluble recombinant oviductin was successfully purified to homogeneity. Full length recombinant oviductin was expressed partially in soluble form, where as the chitinase-like and mucin-like domains of oviductin were expressed in insoluble form and aggregating to form inclusion bodies at both 37 and 16 °C induction temperatures.
Proteomics and Structural Biology Lab, Animal Biotechnology Centre, National Dairy Research Institute, Karnal 132001, India.
Jagadeesh
J
Janjanam
Surender
S
Singh
Suman
S
Choudhary
Mangottil A
MA
Pradeep
Sudarshan
S
Kumar
A
A
Kumaresan
Subrata K
SK
Das
Jai K
JK
Kaushik
Ashok K
AK
Mohanty
D000177:Acrosome / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002020:Buffaloes; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli; D005187:Fallopian Tubes / Q000201:enzymology; D005260:Female; D015870:Gene Expression; D006023:Glycoproteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology; D007668:Kidney / Q000201:enzymology; D008297:Male; D008321:Mammary Glands, Animal / Q000201:enzymology; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D012697:Serine Endopeptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology; D013094:Spermatozoa / Q000502:physiology; D013737:Testis / Q000201:enzymology
D018076:DNA, Complementary; D006023:Glycoproteins; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; C074599:oviductin