title: Bioactivity studies of Huh-7 cells derived human epidermal growth factor expressed in Pichia pastoris.
abstract: Previously, we have reported cloning of human epidermal growth factor gene from Huh-7 cells and its extracellular expression in Pichia pastoris. The presented work is a detailed report regarding molecular characterization of Huh-7 cells-derived hEGF expressed in Pichia pastoris with special reference to its glycosylation profiling and bioactivity studies. Densitometric scanning of SDS-PAGE separated extracellular proteins from hEGF recombinant Pichia pastoris strain indicated that about 84% of the extracellular proteins were glycosylated. Size exclusion chromatography using Superdex 75 prep grade column was successfully utilized to separate fractions containing glycosylated and non-glycosylated extracellular proteins. In dot blot assay, hEGF was detected in both glycosylated and non-glycosylated fractions. Bioactivity assays revealed that both glycosylated and non-glycosylated fractions were bioactive as determined by cell viability assay. It was also observed that hEGF present in non-glycosylated fraction was relatively more bioactive than hEGF present in glycosylated fraction.
authors.affiliation: a National Centre of Excellence in Molecular Biology , University of the Punjab , Lahore , Pakistan.
authors.forename: Mohsin Ahmad
authors.initials: MA
authors.lastname: Khan
authors.affiliation: a National Centre of Excellence in Molecular Biology , University of the Punjab , Lahore , Pakistan.
authors.forename: Nadeem
authors.initials: N
authors.lastname: Ahmed
authors.affiliation: a National Centre of Excellence in Molecular Biology , University of the Punjab , Lahore , Pakistan.
authors.forename: Muhammad Islam
authors.initials: MI
authors.lastname: Khan
authors.affiliation: a National Centre of Excellence in Molecular Biology , University of the Punjab , Lahore , Pakistan.
authors.forename: Ahmad Usman
authors.initials: AU
authors.lastname: Zafar
authors.affiliation: a National Centre of Excellence in Molecular Biology , University of the Punjab , Lahore , Pakistan.
authors.forename: Saad
authors.initials: S
authors.lastname: Tahir
authors.affiliation: a National Centre of Excellence in Molecular Biology , University of the Punjab , Lahore , Pakistan.
authors.forename: Muhammad Sohail
authors.initials: MS
authors.lastname: Anjum
authors.affiliation: a National Centre of Excellence in Molecular Biology , University of the Punjab , Lahore , Pakistan.
authors.forename: Muhammad
authors.initials: M
authors.lastname: Ali
authors.affiliation: a National Centre of Excellence in Molecular Biology , University of the Punjab , Lahore , Pakistan.
authors.forename: Faidad
authors.initials: F
authors.lastname: Khan
authors.affiliation: a National Centre of Excellence in Molecular Biology , University of the Punjab , Lahore , Pakistan.
authors.forename: Tayyab
authors.initials: T
authors.lastname: Husnain
mesh_terms: D000273:Adipose Tissue / Q000166:cytology / Q000187:drug effects / Q000276:immunology; D000818:Animals; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D003001:Cloning, Molecular; D004815:Epidermal Growth Factor / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D015870:Gene Expression; D006031:Glycosylation; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000276:immunology; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D061251:Primary Cell Culture; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology
chemical_list: D011994:Recombinant Proteins; D004815:Epidermal Growth Factor
keywords: Pichia pastoris; epidermal growth factor; glycosylation