Study on the production of recombinant L-asparaginase in Escherichia coli to inhibit cancer cell lines and treatment of acute lymphoblastic leukemia

Theoretical results:

• Construction, expression and purification of rASPG: L-asparaginase was expressed in E. coli BL21(DE3) with high specific activity of 3.2 IU/mg protein. In addition, L-asparaginase was successfully expressed in P. pastoris SMD1168/pPaspg with specific activity of L- asparaginase reached 125 IU/mg protein.
• Characteristic of rASPG: Temperature and pH optimum for rASPG was 45⁰C and 7.5, respectively. The activity of enzyme was enhanced by Mn2+, Pb2+, Mg2+, Ba2+ and inhibited by EDTA, Na+, K+, Fe3+, Ca2+, Ni+, Al3+, Cu2+, Hg2+.
• Study on the production and purification of recombinant L-asparaginase in the Lab scale with expression yields rASPG reached 3.5 mg/g cells. The purified rASPG exhibited L- asparaginase activity, with purify reached 99% by Dolphin 1D solfwave and on SDS-PAGE with molecular mass of 37 kDa.
• rASPG production exhibited cytotoxicity activity tumor cell lines in vitro models such as: SP2/0-Ag14, P3X63Ag8 with IC50 value = 41.67 g/ml and IC50 value = 48.09 g/ml, respectively. In addition, rASPG exhibited no toxicity activity on leukemia cells HL-60 and P388 with IC50 value > 41.67 µg/ml.
• In addition, rASPG exhibited no toxicity to fibroblast cells of mouse (IC50 value > 50 µg/ml) at all concentration rASPG (0,4-50 µg/ml).

Applied results:
The obtained results will orient production and application of potentially to development rASPG for treatment of leukemia acute lymphoblastic leukemia.

• Study on the production of recombinant L-asparaginase in Escherichia coli on
• Determination of inhibited cancer cell lines and treatment of acute lymphoblastic leukemia

- Scientific papers in referred journals (list):
1. Tien Cuong Nguyen, Thi Tuyen Do, Thi Hien Trang Nguyen, Dinh Thi Quyen 2014. “Expression, purification and evaluation of recombinant L-asparaginase in mehthylotrophic yeast Pichia pastoris”J. Viet. Env. 6(1-3): 288-292.
2. Do Thi Tuyen, Nguyen Tien Cuong, Nguyen Thi Hien Trang, Quyen Dinh Thi, 2013 “Cloning expression and purification of gene encoding L- asparaginae in Pichia pastoris GS115” The 3rd Academic Conference on Natural Science for Master and PhD Students From Asean countries. 11-15, November 2013, Phnom penh, Cambodia; 279-289.
3. Nguyen Thi Hien Trang, Do Thi Tuyen, Quyen Dinh Thi, 2013. “Expression of Asparaginase gene in E.coli” The 3rd Academic Conference on Natural Science for Master and PhD Students From Asean countries. 11-15, November 2013, Phnom penh, Cambodia; 307- 316
4. Nguyen Thi Hien Trang, Do Thi Tuyen, Quyen Dinh Thi, 2014 ”Expression, purification recombination L- asparaginase in E. coli, and initially assessed the biological activity of L- asparaginase in THP1 leukemia cell line. J. Viet. Med.; 421; 13-17.
5. 01 manuscript of Allergy, asthma & Clinical Immunology (AACI): Nguyen Thi Hien Trang, Do Thi Tuyen, Nguyen Tien Cuong, Nguyen Sy Le Thanh, Quyen Dinh Thi, 2014 “Expression optimization, characterization and anti-cancer activity of recombinant L- asparaginase II in E.coli ”.
- Technological products (describe in details: technical characteristics, place):

• Technology for production of 1 g rASPG, which exhibited L- asparaginase activity, with purify reached 99% by Dolphin 1D solfwave and on SDS-PAGE (Products are stored at Enzyme of Biotechnology department, IBT, VAST);
• rASPG production exhibited cytotoxicity activity tumor cell lines in vitro models such as: SP2/0-Ag14, P3X63Ag8 with IC50 value = 41.67 µg/ml and IC50 value = 48.09 µg/ml, respectively. In addition, rASPG exhibited no toxicity activity on leukemia cells HL-60 and P388 with IC50 value > 41.67 µg/ml;
• L-asparaginase toxicity is not shown for NIH/3T3 cells at all concentration of rASPG (0,4-50 µg/ml) with IC50 values> 50 µg/ml.

- Other products (if applicable):
1. Conference papers: five papers; 01 manuscript of Allergy, asthma & Clinical Immunology (AACI).
2. Research training:

• 01 MSc and 01 BSc, who successfully defended the thesises in 2014;
• 01 PhD student, who is studying the thesises, stages: 2013-2017.

The funding should be continued for this project to carry out the researches on:
- Study on the production of recombinant L-asparaginase in Pichia
- Testing anti-cancer activity of recombinant L- asparaginase in Pichia”