Extracellular expression and affinity purification of L-asparaginase from E. chrysanthemi in E. coli

Abstract

L-asparaginases (E.C.3.5.1.1, L-ASNases) are successfully used as anti-neoplastic agents in the chemotherapy of acute lymphoblastic leukemia (ALL) and therefore are of high interest for the medical and scientific community. In the present study we report the recombinant, extracellular expression and affinity purification of L-asparaginase from Erwinia chrysanthemi 3937 (ErL-ASNase) in E. coli. Recombinant E. coli strains were screened for their ability to express and translocate ErL-ASNase to the culture medium. The strain E. coli Rosetta (DE3) exhibited the highest extracellular expression levels among all the strains tested and it was chosen for further optimization and the development of purification protocol. Affinity adsorbents with immobilized L-Asn, L-Asp and L-Glu were synthesized by solid-phase chemistry and evaluated for their ability to bind and purify ErL-ASNase directly from the culture medium. The affinity adsorbent with immobilized L-Asp (L-Asp-Sepharose CL-6B) showed the highest purifying ability for ErL-ASNase. Adsorption equilibrium studies revealed that the adsorption of ErL-ASNase follows Langmuir isotherm with KD = 0.21 μM and maximum binding capacity 4.7 mg enzyme/g moist wet adsorbent. This affinity adsorbent was used for the development of one-step purification protocol. The proposed protocol is simple, fast, gentle and afforded ErL-ASNase with high purity and yield. We show that the recombinant expression of ErL-ASNase in E. coli results in the secretion to the culture medium due to the presence of its natural peptide leader at the N-terminus. We developed an L-Asp-based affinity adsorbent which allowed the purification of the enzyme in one step, achieving high purity levels. This approach is advantageous over the other conventional tag-based purification methods, which require additional treatment steps for the cleavage and isolation of the affinity tags. Overall, the strategy employed for expression and purification of this protein drug uses green chemistry principles allowing the reduction of processing time and purification steps, making the approach more sustainable and attractive.