What is MTX selection?

The methotrexate (MTX) selection system is used to select CHO cells producing the protein of interest. MTX, a drug similar to folate, binds to DHFR, thereby inhibiting the production of tetrahydrofolate, which is necessary for the de-novo synthesis of purines and pyrimidines (Goeddel, 1990).

What is dihydrofolate reductase catalysis?

E. coli dihydrofolate reductase (ecDHFR) serves as a classic model of acid–base catalysis. DHFR catalyzes the NADPH-dependent reduction of 7,8-dihydrofolate (DHF) to 5,6,7,8-tetrahydrofolate (THF) for nucleotide and amino acid synthesis during cell proliferation.

What happens when dihydrofolate reductase is inhibited?

The inhibition of dihydrofolate reductase results in depletion of intracellular pools of reduced folates. The lack of reduced folates impairs synthesis of purine nucleotides, thymidylate, and certain amino acids, which can lead to cell death. First dihydrofolate reductase inhibitor, aminopterin (28.1.

What are dihydrofolate reductase inhibitors?

A substance that can build up in cancer cells and block them from using folate. Folate is a nutrient that rapidly dividing cells need to make DNA. Blocking folate use helps keep cancer cells from growing and may kill them.

What is DHFR selection system?

Gene amplification is achieved by drug resistance. In this case (DHFR-MTX selection system) CHO cells are subjected to increasing concentration of MTX making them resistance to MTX via gene amplification. In transfected cells the recombinant DNA consists of the GOI closely linked to the gene for DHFR.

Why is methotrexate used in cell culture?

Low concentrations of MTX are then used to amplify the transfected genes for increased protein expression. A single cell cloning protocol is also described. This can be used after each stage of MTX amplification to isolate high-expressing clones that are also consistent producers over longer culture periods.

Is folic acid a substrate for dihydrofolate reductase?

Folic Acid as Substrate for DHFR. Due to the slow rate of DHFR with FA, activity with this substrate was measured only by the HPLC method. The rate of THF formation by DHFR from rat liver with FA as substrate was 850 times slower than with 7,8-DHF (Table 2).

Which drug inhibits the dihydrofolate reductase enzyme?

Methotrexate. Methotrexate is a dihydrofolate reductase inhibitor used in the treatment of leukemia, lymphomas (including central nervous system lymphoma), choriocarcinoma, breast cancer, osteosarcoma, and leptomeningeal metastases.

Which drug inhibits dihydrofolate reductase and the subsequent production of purines?

Trimethoprim (TMP), (2,4-diamino-5-(3′,4′,5′-trimethoxybenzyl)pyrimidine) is the well-known dihydrofolate reductase inhibitor and one of the standard antibiotics used in urinary tract infections (UTIs).