Using Recombinant Escherichia Coli Production Of 2,3 - Diphosphoglycerate Acid Research

2,3-BPG(2,3-Bisphosphoglycerate)is an isomer of the 1,3-BPG(1,3-Bisphosphoglycerate) which acts as an intermediate in the glycolysis pathway. It can be catalyze by the exclusive enzyme - BPGM(Bisphosphoglycerate Mutase,old name Diphosphoglycerate Mutase,DPGM)that merely exists in erythrocyte and placenta.2,3-BPG was first found in 1925,but not until 1965 had its function to be cleared by Benesch. Cause 2,3-BPG acts as an allosteric model and combines with the hemoglobin equally in amount. Thus it plays a critical role in adjusting the affinity between O2 and hemoglobin. When blood flows pass tissues, the existence of high amount of 2,3-BPG would extraordinarily increase the release of O2 from hemoglobin to tissues which make 2,3-BPG becomes a star molecular in preventing and curing the high altitude syndrome, improving the body function of atheletes. However, nowadays there's no records on synthesize of 2,3-BPG and clearly not a considerable choice to restore from blood. In the paper, we establish a recombinant E.coli express system to produce 2,3-BPG.We transform the BL21(DE3) E.coli strain with an express plasmid that contains human BPGM gene and induce with IPTG. Through providing glucose into the LB medium, we use the glycolysis pathway in E.coli to produce 2,3-BPG. Cause BPGM acts not only as 2,3-BPG synthesis enzyme but also performs phosphate enzyme activity especially with the existence of Cl- divides 2,3-BPG into 3-PGA and phosphate. Malachite green agent is used to determine the quantity of phosphate, after compared with standard phosphate content curve we can calculate the amount of 2,3-BPG in the sample.We found an optimal glucose concentration as 10 g/L, an appropriate inducing time as 4 hours. Also, adding Tween 80 to final concentration 0.5% greatly help 2,3-BPG excretes into LB and can be directly restore from the LB after centrifugal process.