title: In vitro refolding with simultaneous purification of recombinant human parathyroid hormone (rhPTH 1-34) from Escherichia coli directed by protein folding size exclusion chromatography (PF-SEC): implication of solution additives and their role on aggregates and renaturation.
abstract: Recombinant proteins are frequently hampered by aggregation during the refolding and purification process. A simple and rapid method for in vitro refolding and purification of recombinant human parathyroid hormone (rhPTH 1-34) expressed in Escherichia coli with protein folding size exclusion chromatography (PF-SEC) was developed in the present work. Discrete effects of potential solution additives such as urea, polypolyethylene glycol, proline, and maltose on the refolding with simultaneous purification of rhPTH were investigated. The results of individual additives indicated that both maltose and proline had remarkable influences on the efficiency of refolding with a recovery yield of 65 and 66% respectively. Further, the synergistic effect of these additives on refolding was also explored. These results demonstrate that the additive combinations are more effective for inhibiting protein aggregation during purification of rhPTH in terms of recovery yield, purity, and specific activity. The maltose and proline combination system achieved the highest renatured rhPTH having a recovery yield of 78%, a purity of ≥99%, and a specific activity of 3.31 × 10(3) cAMP pM/cell respectively, when compared to the classical dilution method yield (41%) and purity (97%). In addition, the role of maltose and proline in a combined system on protein aggregation and refolding has been explained. The molecular docking (in silico) scores of maltose (-10.91) and proline (-9.0) support the in vitro results.
authors.affiliation: Centre for Bioprocess Technology, Department of Biotechnology, K L E F University, Green Fields, Vaddeswaram, Guntur, 522 502, Andhra Pradesh, India.
authors.forename: Sandeep
authors.initials: S
authors.lastname: Vemula
authors.affiliation: Department of Pharmacology, Kakatiya University, Vidyaranyapuri, Hanamkonda, Warangal, Telangana, 506009, India.
authors.forename: Sushma
authors.initials: S
authors.lastname: Vemula
authors.affiliation: Centre for Bioprocess Technology, Department of Biotechnology, K L E F University, Green Fields, Vaddeswaram, Guntur, 522 502, Andhra Pradesh, India.
authors.forename: Akshay
authors.initials: A
authors.lastname: Dedaniya
authors.affiliation: Centre for Bioprocess Technology, Department of Biotechnology, K L E F University, Green Fields, Vaddeswaram, Guntur, 522 502, Andhra Pradesh, India. rsr@kluniversity.in.
authors.forename: Srinivasa Reddy
authors.initials: SR
authors.lastname: Ronda
mesh_terms: D002850:Chromatography, Gel / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D062105:Molecular Docking Simulation; D010281:Parathyroid Hormone / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D066329:Protein Aggregates; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: C470125:PTH protein, human; D010281:Parathyroid Hormone; D066329:Protein Aggregates; D011994:Recombinant Proteins
keywords: PFLC; Purity; Recovery yield; Size exclusion chromatography; Specific activity; rhPTH