Large‐scale purification and characterization of recombinant human stem cell factor in Escherichia coli

Abstract

The pharmacological importance of recombinant human stem cell factor (rhSCF) has increased the demand to establish effective and large-scale production and purification processes. A good source of bioactive recombinant protein with capability of being scaled-up without losing activity has always been a challenge. The objectives of the study were the rapid and efficient pilot-scale expression and purification of rhSCF. The gene encoding stem cell factor (SCF) was cloned into pBV220 and transformed into Escherichia coli. The recombinant SCF was expressed and isolated using a procedure consisting of isolation of inclusion bodies (IBs), denaturation, and refolding followed by chromatographic steps toward purification. The yield of rhSCF reached 835.6 g/20 L, and the expression levels of rhSCF were about 33.9% of the total E. coli protein content. rhSCF was purified by isolation of IBs, denaturation, and refolding, followed by SP-Sepharose chromatography, Source 30 reversed-phase chromatography, and Q-Sepharose chromatography. This procedure was developed to isolate 5.5 g of rhSCF (99.5% purity) with specific activity at 0.96 × 10 IU/mg, endotoxin levels of pyrogen at 1.0 EU/mg, and bacterial DNA at 10 ng/mg. Pilot-scale fermentations and purifications were set up for the production of rhSCF that can be upscaled for industry.

Three Novel Escherichia coli Vectors for Convenient and Efficient Molecular Biological Manipulations

“…However, specific E. coli strains expressing T7 polymerase are required and inducer concentrations (IPTG, Arabinose) may need to be tuned to mitigate potential negative effects associated with the high viral polymerase activity . Temperature-inducible protein expression vectors (e.g., pBV220, pSW2, pKF396M-5) are convenient because only shifting the culture temperature is required. − However, most established vectors are only available for sticky-end cloning with the limitations described above. Also, a fixed induction temperature is recommended for protein expression which may be problematic in some cases. − …”

Pengaruh Jumlah Inokulum Sel Inang Bakteri E.coli BL21 (DE3) pLysS dan Waktu Overekspresi pada Produksi Protein Rekombinan Fim-C Salmonella typhi

“…Analisis literatur menunjukkan bahwa banyak hal yang harus diperhatikan dalam produksi rekombinan protein sebagai vaksin, atau sebagai tujuan terapeutik dan diagnosis. Salah satu sukses yang paling utama dalam aplikasi molekul biosimilar tersebut adalah bila berhasil mengembangkan produk tersebut dalam skala besar (Brown et al, 2014 ;Bustos et al, 2016 ;Chen et al, 2016).…”

Novel Bacterial Production of Two Different Bioactive Forms of Human Stem-Cell Factor

“…However, difficulties remain in the use of this system and in the comparatively high cost. For the more economic and efficient production of hSCF164, heterologous expression using Escherichia coli has been adopted [13,[25][26][27][28][29][30]. However, hSCF164 expressed in E. coli resulted in denatured proteins and inclusion bodies.…”