Simple procedure applying lactose induction and one‐step purification for high‐yield production of rhCIFN

Abstract

Recombinant consensus interferon (CIFN) is a therapeutic protein with molecular weight of 19.5 kDa having broad spectrum antiviral activity. Recombinant human CIFN (rhCIFN) has previously been expressed in Escherichia coli using isopropyl-β-d-thiogalactopyranoside (IPTG), a non-metabolizable and expensive compound, as inducer. For economical and commercial-scale recombinant protein production, it is greatly needed to increase the product yield in a limited time frame to reduce the processing cost. To reduce the cost of production of rhCIFN in E. coli, induction was accomplished by using lactose instead of IPTG. Lactose induction (14 g/L) in shake flask experiment resulted in higher yield as compared with 1 mM IPTG. Finally, with single-step purification on DEAE sepharose, 150 mg/L of >98% pure rhCIFN was achieved. In the present study, an attempt was made to develop a low cost process for producing quality product with high purity. Methods devised may be helpful for pilot-scale production of recombinant proteins at low cost.

The Effects of Lactose Induction on a Plasmid-Free E. coli T7 Expression System

“…Previously, only plasmid-based expression systems had been subject to studies investigating lactose as an inducer for recombinant protein production [23,36,37]. In those studies, beneficial effects on productivity, cell fitness, viability, and soluble product formation had been reported [21,22,[38][39][40].…”

“…For plasmid-based systems it has been reported that lactose induction provided the advantages of increased soluble product formation and cell fitness [19][20][21][22]. However, as the high specific lactose uptake rates of the GI strain clearly seemed toxic to the cells, we wanted to check if perhaps IPTG induction was beneficial for this strain.…”

“…Nevertheless, it has been reported that IPTG puts a high metabolic burden on the cells, decreases the amount of soluble recombinant protein, and exacerbates substrate toxicity [17][18][19]. One way to tackle these adverse effects is to use lactose as inducer, which has been shown to yield in similar if not higher product titers and to increase soluble product formation and cell fitness, enabling longer production times [19][20][21][22]. Additionally, lactose is cheap and non-toxic.…”

Mechanistic platform knowledge of concomitant sugar uptake in Escherichia coli BL21(DE3) strains

“…However, IPTG is known to put a high metabolic burden on E. coli 910 and is often associated with the generation of misfolded protein aggregates, called inclusion bodies1112. In contrast, the alternative inducer lactose has been shown to favour the production of soluble product and trigger enhanced productivity13141516. Even though lactose metabolism is topic of some recent studies17, lactose is scarcely used in biochemical engineering since induction entails the challenge of continuous supply of the disaccharide as it gets metabolized by E. coli .…”

Enhancing recombinant interleukin‐6 production yield by fermentation optimization, two‐step denaturing, and one‐step purification

“…The refolded protein of the recombinant interleukin‐6 was purified on the AKTA system (GE Healthcare, USA) by using the ion exchange XK26/10 column packed with DEAE Sepharose CL6B. Prior to loading the refolded protein on the column, l ‐arginine and other salts were removed by applying the diafiltration procedure as described . The diafilterd protein was loaded onto the preequilibrated column with 20 cv of the buffer (20 mM sodium phosphate pH 7, 0.01% Tween 20) at the flow rate of 3 mL/Min.…”