Overexpression in Escherichia coli and purification of human fibroblast growth factor (FGF-2)

Abstract

Basic fibroblast growth factor (FGF-2) is a member of a large family of structurally related proteins that affect the growth, differentiation, migration, and survival of many cell types. The human FGF-2 gene (encoding residues 1-155) was synthesized by PCR from 20 oligonucleotides and cloned into plasmid pET-32a. A high expression level (1 g/liter) of a fused protein thioredoxin/FGF-2 was achieved in Escherichia coli strain BL21(DE3). The fusion protein was purified from the soluble fraction of cytoplasmic proteins on a Ni-NTA agarose column. After cleavage of the thioredoxin/FGF-2 fusion with recombinant human enteropeptidase light chain, the target protein FGF-2 was purified on a heparin-Sepharose column. The yield of FGF-2 without N- and C-terminal tags and with high activity was 100 mg per liter of cell culture. Mutations C78S and C96S in the amino acid sequence of the protein decreased FGF-2 dimer formation without affecting its solubility and biological activity.

Expression and Purification of Recombinant Human Basic Fibroblast Growth Factor Fusion Proteins and Their Uses in Human Stem Cell Culture

“…0.1-0.2 ng hbFGF ml -1 ) than the commercially available hbFGF (ED 50 27 p M ). This finding was consistent with previous reports of the mitogenic activity of recombinant hbFGF, which had an ED 50 of approximately 0.15-3 ng ml -1 [Gasparian et al, 2009;Song et al, 2013]. Moreover, the purified hbFGF produced in this study showed higher activity when compared to the activity of the commercial hb-FGF.…”

“…However, we found that only approximately 20-60% of the expressed fusion proteins bind to the Ni 2+ IMAC. This result was inconsistent with previous studies by Rassouli et al [2013] and Gasparian et al [2009]. Several purification processes have been established for the production of recombinant hbFGF from E. coli , including single-step purification with heparin affinity chromatography or immobilized metal ion affinity chromatography, combination of cation exchange column chromatography and heparin affinity chromatography, and combination of continuous bed or expanded bed chromatography and heparin affinity chromatography.…”

“…Several hosts, such as E. coli , Pichia pastoris , insect cells, Saccharomyces cerevisiae , Bacillus subtilis , soybean seeds, silkworms (Bombyx mori L.) and rice seeds, have been used for expressing recombinant hbFGF with yields of approximately 1-100 mg of protein per liter of cell culture [Ding et al, 2006;Gasparian et al, 2009;Hills and Crane-Robinson, 1995;Kwong et al, 2013;Mu et al, 2008;Rassouli et al, 2013;Song et al, 2013;Squires et al, 1988;Wu et al, 2001]. E. coli is the most favorable expression host for the expression of hbFGF because of the availability of versatile vector systems, host strains that facilitate high-level expression of recombinant proteins and rapid growth on inexpensive media.…”

A simple method for production and purification of soluble and biologically active recombinant human leukemia inhibitory factor (hLIF) fusion protein in Escherichia coli

“…Many studies have successfully used Escherichia coli to produce several human growth factors and cytokines, such as interleukin-1 (Furutani et al, 1985), interleukin-4 (van Kimmenade et al, 1988), epidermal growth factor (Shimizu et al, 1991;Abdull Razis et al, 2008), fibroblast growth factor (Squires et al, 1988;Gasparian et al, 2009) and LIF (Gearing et al, 1989;Samal et al, 1995;Tomala et al, 2010) for therapeutic and clinical purposes. Although, recombinant human LIF (hLIF) is available and has been successfully produced in E. coli expression systems, they still require many steps of downstream processing.…”

Peculiarities of Gene Transfer into Mesenchymal Stem Cells

“…Biol. Sciences, Laboratory of Protein Engineering, Institute of Bioorganic Chemistry, Russian Academy of Sciences) [5]. In 14 days, fi broblast colonies were stained with 0.1% crystal violet in 20% methanol and counted under an inverted microscope (the formations consisting of at least 50 cells were considered colonies).…”