title: Expression and rapid purification of recombinant biologically active ovine growth hormone with DsbA targeting to Escherichia coli inner membrane.
abstract: This study shows expression of recombinant ovine growth hormone (roGH) and targeting to the inner membrane using signal sequence, DsbA, in Escherichia coli (E. coli) cell. Factors such as temperature, IPTG induction, and expression conditions were studied and show diverse optical density with different media compositions. The optimum expression level of roGH in terrific broth medium was at 25 °C on induction with 20 μM IPTG in early logarithmic phase. SDS-PAGE analysis of expression and subcellular fractions of recombinant constructs revealed the translocation of roGH to the inner membrane of E. coli with DsbA signal sequence at the N terminus of roGH. The protein was easily solubilized by 40 % acetonitrile with ~90 % purity and was identified by Western blot, and analysis on MALDI-TOF/TOF confirmed a size of 21,059 Da. Relatively high soluble protein yield of 65.3 mg/L of roGH was obtained. The biological function of roGH was confirmed by HeLa cell line proliferation. This is the first study describing achievement of biologically active soluble roGH targeted to the inner membrane of E. coli and rapid purification with high yield.
authors.affiliation: School of Biological Sciences, University of the Punjab, Lahore, Pakistan.
authors.forename: Faiza Gul
authors.initials: FG
authors.lastname: Durrani
authors.forename: Roquyya
authors.initials: R
authors.lastname: Gul
authors.forename: Saima
authors.initials: S
authors.lastname: Sadaf
authors.forename: Muhammad Waheed
authors.initials: MW
authors.lastname: Akhtar
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D049109:Cell Proliferation / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel; D004847:Epithelial Cells / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics*; D015870:Gene Expression; D006041:Goats; D013006:Growth Hormone / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D006801:Humans; D008970:Molecular Weight; D019704:Protein Disulfide-Isomerases / Q000235:genetics*; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature; D013997:Time Factors; D015533:Transcriptional Activation
chemical_list: D029968:Escherichia coli Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D013006:Growth Hormone; D019704:Protein Disulfide-Isomerases; C526648:dsbA protein, E coli