Production of red fluorescent protein (mCherry) in an inducible E. coli expression system in a bioreactor, purification and characterization

Yıl 2019, Cilt: 3 Sayı: 1, 20 - 25, 15.04.2019

Hülya Kuduğ Bahadır Ataman Rizvan İmamoğlu Duygu Düzgün İsa Gökçe

Öz

New and improved genetic
engineered variants of fluorescent proteins (FPs) have become useful tools for bioimaging in biomedical
researches. Red fluorescent proteins (RFPs) first derived from the sea anemone Discosoma show high performance in vivo labeling and imaging. mCherry is
a member of RFPs which has very high
photostability, resistant to photo bleaching and rapid maturation. These
advantages ensure that mCherry
can be successfully fused to many proteins and widely used for quantitative
imaging techniques. In this study, the constructed recombinant plasmid
pBADCherry was expressed in Escherichia
coli BL21(AI) then culture conditions, inducer concentration and induction
time were optimized. Results of sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
analysis demonstrated that 5 hours induction at 0.04%
of arabinose concentration was optimal for the highest mCherry yield. The expression of hexa histidine-tagged
(6xHis) recombinant mCherry was induced by arabinose and purification performed
using nickel (Ni²⁺) affinity chromatography. High throughput
expression of 81 mg fluorescent protein from a liter of E. coli culture carried out in bioreactor.

Anahtar Kelimeler

Bioreactor, E.coli, Fluorescent Proteins, mCherry, pBAD

Kaynakça

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