title: Optimization of expression and purification of HSPA6 protein from Camelus dromedarius in E. coli.
abstract: The HSPA6, one of the members of large family of HSP70, is significantly up-regulated and has been targeted as a biomarker of cellular stress in several studies. Herein, conditions were optimized to increase the yield of recombinant camel HSPA6 protein in its native state, primarily focusing on the optimization of upstream processing parameters that lead to an increase in the specific as well as volumetric yield of the protein. The results showed that the production of cHSPA6 was increased proportionally with increased incubation temperature up to 37 °C. Induction with 10 μM IPTG was sufficient to induce the expression of cHSPA6 which was 100 times less than normally used IPTG concentration. Furthermore, the results indicate that induction during early to late exponential phase produced relatively high levels of cHSPA6 in soluble form. In addition, 5 h of post-induction incubation was found to be optimal to produce folded cHSPA6 with higher specific and volumetric yield. Subsequently, highly pure and homogenous cHSPA6 preparation was obtained using metal affinity and size exclusion chromatography. Taken together, the results showed successful production of electrophoretically pure recombinant HSPA6 protein from Camelus dromedarius in Escherichia coli in milligram quantities from shake flask liquid culture.
authors.affiliation: Protein Research Chair, Department of Biochemistry, College of Science, King Saud University, PO Box 2455, Riyadh 11451, Saudi Arabia.
authors.forename: Ajamaluddin
authors.initials: A
authors.lastname: Malik
authors.affiliation: Protein Research Chair, Department of Biochemistry, College of Science, King Saud University, PO Box 2455, Riyadh 11451, Saudi Arabia.
authors.forename: Abdulrahman M
authors.initials: AM
authors.lastname: Alsenaidy
authors.affiliation: Genome Research Chair, Department of Biochemistry, College of Science, King Saud University, PO Box 2455, Riyadh 11451, Saudi Arabia; Biochemistry Department, Faculty of Science, Ain-Shams University, Abbassia 11381, Cairo, Egypt.
authors.forename: Mohamed
authors.initials: M
authors.lastname: Elrobh
authors.affiliation: Department of Basic Sciences, College of Science and Health Professions, King Saud Bin Abdulaziz University for Health Sciences, PO Box 3660, Riyadh 11426, Saudi Arabia.
authors.forename: Wajahatullah
authors.initials: W
authors.lastname: Khan
authors.affiliation: Genome Research Chair, Department of Biochemistry, College of Science, King Saud University, PO Box 2455, Riyadh 11451, Saudi Arabia.
authors.forename: Mohammed S
authors.initials: MS
authors.lastname: Alanazi
authors.affiliation: Protein Research Chair, Department of Biochemistry, College of Science, King Saud University, PO Box 2455, Riyadh 11451, Saudi Arabia; Genome Research Chair, Department of Biochemistry, College of Science, King Saud University, PO Box 2455, Riyadh 11451, Saudi Arabia.
authors.forename: Mohammad D
authors.initials: MD
authors.lastname: Bazzi
keywords: 2× LB, double strength Luria–Bertani; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; Expression optimization; FPLC, fast protein liquid chromatography; Fast protein liquid chromatography; Heat shock protein; Hsp70; IPTG, isopropyl β-d-1-thiogalactopyranoside; LB, Luria–Bertani; Molecular chaperone; NB, nutrient broth; Ni–NTA, nickel–nitrilotriacetic acid; OD600, optical density at 600 nm; PMSF, phenylmethylsulfonyl fluoride; Recombinant; TB, terrific broth; amp, ampicillin; rpm, rotations per minute