Optimization of Buffer Additives for Efficient Recovery of hGM-CSF from Inclusion Bodies Using Response Surface Methodology.

Iran J Pharm Res

Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Research Center, Faculty of Pharmacy, Isfahan University of Medical Sciences, Isfahan, Iran.

Published: January 2020

Overexpression of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) by leads to formation of insoluble and inactive proteins, inclusion bodies. The aim of this study was to improve recovery of biologically active hGM-CSF from inclusion bodies. The effect of types, concentrations and pHs of denaturing agents and addition of reducing agents on the yield of inclusion bodies solubilization was evaluated. Next, various conditions were evaluated for refolding hGM-CSF using a two-step design of experiment (DOE) including primary screening by factorial design, and then optimization by response surface design. It was found that hGM-CSF inclusion bodies can be efficiently solubilized with 4 M urea and 4 mM β-mercaptoethanol, pH = 9. A response surface quadratic model was employed to predict the optimum refolding conditions and the accuracy of this model was confirmed by high value of R (0.99) and F-value of 0.64. DOE results revealed that sorbitol (0.235 M), imidazole (97 mM), and SDS (0.09%) would be the optimum buffer additives for refolding of hGM-CSF. Following refolding studies, the obtained protein was subjected to circular dichroism which confirmed correct secondary structure of the refolded hGM-CSF. The refolded hGM-CSF exhibited reasonable biological activity compared with standard protein. The approach developed in this work can be important to improve the refolding of other proteins with similar structural features.

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7758011PMC
http://dx.doi.org/10.22037/ijpr.2020.1101169DOI Listing

Publication Analysis

Top Keywords

inclusion bodies
20
hgm-csf inclusion
12
response surface
12
buffer additives
8
hgm-csf
8
refolding hgm-csf
8
refolded hgm-csf
8
inclusion
5
bodies
5
refolding
5

Similar Publications

Dectin-1 (CLEC7A), a C-type lectin-like receptor that recognizes β-1,3 glucans, has a key role in the innate immune system. While the lectin domain of mouse Dectin-1 has been solubilized and refolded from inclusion bodies in Escherichia coli, similar refolding of the human Dectin-1 lectin domain is hindered by the formation of misfolded multimers with aberrant intermolecular disulfide bonds. The aim of this study was to develop a method for the large-scale production of the human Dectin-1 lectin domain.

View Article and Find Full Text PDF

Detection of Tilapia parvovirus in farm-reared tilapia in India and its isolation using fish cell lines.

In Vitro Cell Dev Biol Anim

January 2025

Aquatic Animal Health Laboratory, PG & Research Department of Zoology, C. Abdul Hakeem College, (Affiliated to Thiruvalluvar University), Melvisharam, Tamil Nadu, India.

Tilapia parvovirus (TiPV) is an emerging viral pathogen and responsible for severe economic loss in tilapia culture production. Lethargic, cutaneous haemorrhages; ocular lesions; discolouration of gill and cloudy eye and exophthalmia are common symptoms of TiPV. The TiPV-suspected tilapia fish were collected from grow-out ponds situated in different parts of Tamil Nadu, India, and screened for TiPV by PCR.

View Article and Find Full Text PDF

Unlabelled: Since the inception of transplantation, it has been crucial to ensure that organ or tissue donations are made with valid informed consent to avoid concerns about coercion or exploitation. This issue is particularly challenging when it comes to infants and younger children, insofar as they are unable to provide consent. Despite their vulnerability, infants' organs and tissues are considered valuable for biomedical purposes due to their size and unique properties.

View Article and Find Full Text PDF

Distinct subcellular localization of tau and alpha-synuclein in lewy body disease.

Acta Neuropathol Commun

January 2025

Center for Neurodegeneration and Experimental Therapeutics, Department of Neurology, University of Alabama at Birmingham, Birmingham, AL, USA.

Lewy bodies and neurofibrillary tangles, composed of α-synuclein (α-syn) and tau, respectively, often are found together in the same brain and correlate with worsening cognition. Human postmortem studies show colocalization of α-syn and tau occurs in Lewy bodies, but with limited effort to quantify colocalization. In this study, postmortem middle temporal gyrus tissue from decedents (n = 9) without temporal lobe disease (control) or with Lewy body disease (LBD) was immunofluorescently labeled with antibodies to phosphorylated α-syn (p-α-syn), tau phosphorylated at Ser202/Thr205 (p-tau), or exposure of tau's phosphatase-activating domain (PAD-tau) as a marker of early tau aggregates.

View Article and Find Full Text PDF

West Nile virus (WNV) infection is emerging as a disease of public health concern in Kerala, India with recurring outbreaks since 2011. With its tropical climate, biodiversity hot spots of Western ghats, forest cover, plenty of water bodies and bird sanctuaries, Kerala provides an ideal ecological niche for vector breeding and transmission of WNV. In this article, we reflect on the peculiar features of the outbreaks of WNV fever in Kerala and highlight the knowledge gaps, the research priorities and the need for effective control measures.

View Article and Find Full Text PDF

Want AI Summaries of new PubMed Abstracts delivered to your In-box?

Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!