Cloning, expression, and characterization of human brain acetylcholinesterase in Escherichia coli using a SUMO fusion tag

Ceylan H., Erdoğan O.

TURKISH JOURNAL OF BIOLOGY, cilt.41, ss.77-87, 2017 (SCI-Expanded) identifier identifier

  • Yayın Türü: Makale / Tam Makale
  • Cilt numarası: 41
  • Basım Tarihi: 2017
  • Doi Numarası: 10.3906/biy-1602-83
  • Dergi Adı: TURKISH JOURNAL OF BIOLOGY
  • Derginin Tarandığı İndeksler: Science Citation Index Expanded (SCI-EXPANDED), Scopus, TR DİZİN (ULAKBİM)
  • Sayfa Sayıları: ss.77-87
  • Anahtar Kelimeler: Acetylcholinesterase, cloning, recombinant protein, enzyme characterization, N-LINKED GLYCOSYLATION, BIOCHEMICAL FUNDAMENTALS, THERAPEUTIC PROTEINS, RECOMBINANT PROTEINS, COMMERCIAL SYSTEMS, ALZHEIMERS-DISEASE, INHIBITORS, GENE, DIFFERENTIATION, CHOLINESTERASES
  • Atatürk Üniversitesi Adresli: Evet

Özet

The molecular structure of acetylcholinesterase (AChE) attracts interest because of its versatility and significant role in the cholinergic system. The main purpose of the present study was to clone a full-length cDNA sequence of human brain acetylcholinesterase (hAChE) into pET SUMO vector and express it successfully. The integrity of the constructed plasmid was confirmed by cross PCR. This recombinant construct was expressed in Escherichia coli BL21 (DE-3). In this work, we produced hexahistidine (6xHis) tagged fusion protein by isopropyl beta-D-1-thiogalactopyranoside (IPTG) induction and purified using nickel (Ni2+) affinity chromatography. Using anti-His antibody, we detected similar to 90 kDa fusion protein. The expression of the hAChE gene in a microbial host resulted in good biological activity. Using the Ellman method, the recombinant AChE exhibited activity with optima at pH 9.0 glycine-NaOH buffer and room temperature. Kinetic parameters, K (M) and V (max), were determined as 0.63 and 0.69, respectively.