Yang Q H, Wu C L, Lin K, Li L
Shanghai Institute of Biochemistry, Academia Sinica, China.
Protein Expr Purif. 1997 Aug;10(3):320-4. doi: 10.1006/prep.1997.0749.
Expression of chicken and rat liver bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, in Escherichia coli encountered two common problems: the chicken enzyme was liable to proteolysis and the rat enzyme was prone to form inclusion bodies. Reducing the rate of protein synthesis by lowering either growth temperature or isopropyl-beta-D-thiogalactopyranoside (IPTG) concentration alleviated these two problems. Growth at 22 degrees C was optimum for expression of both enzymes. The optimum range of IPTG concentration for expression was 0.1-1 microM for the chicken liver bifunctional enzyme and 10 microM for rat liver enzyme. The components of growth medium also influenced the production. Compared with Luria-Bertani medium, an enriched medium-tryptone-phosphate medium-tripled the production of the active enzymes. Addition of glucose (0.2%) doubled the expression level of active chicken liver enzyme, but reduced the production of active rat liver enzyme to half the maximal level, while the phosphate in tryptone-phosphate medium had no effect on the production of the two enzymes.
鸡和大鼠肝脏双功能酶6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶在大肠杆菌中的表达遇到两个常见问题：鸡源酶易于被蛋白水解，而大鼠源酶易于形成包涵体。通过降低生长温度或异丙基-β-D-硫代半乳糖苷（IPTG）浓度来降低蛋白质合成速率可缓解这两个问题。22℃生长最适合两种酶的表达。鸡肝脏双功能酶表达的IPTG最佳浓度范围为0.1 - 1微摩尔，大鼠肝脏酶为10微摩尔。生长培养基的成分也影响产量。与Luria-Bertani培养基相比，富集培养基胰蛋白胨-磷酸盐培养基使活性酶产量增加两倍。添加葡萄糖（0.2%）使活性鸡肝脏酶的表达水平加倍，但使活性大鼠肝脏酶的产量降至最大水平的一半，而胰蛋白胨-磷酸盐培养基中的磷酸盐对两种酶的产量没有影响。
