~(21)Ala定点突变胰高血糖素及结构与功能变化

 胰高血糖素是由 2 9个氨基酸组成的多肽激素 ,具有促糖元分解的生理功能 ,其拮抗剂有治疗糖尿病病人的潜在应用价值 .在获得重组胰高血糖素基因工程菌基础上 ,利用定点突变技术改造其第 2 1位氨基酸天冬氨酸为丙氨酸 ,并经DNA测序证明胰高血糖素基因发生了点突变 .用IPTG诱导表达后 ,经亲和层析和反相高效液相层析 ,纯化到突变型重组2 1Ala 胰高血糖素 .质谱测定分子量与理论值相符 .利用园二色谱比较重组胰高血糖素和突变的2 1Ala 胰高血糖素在TFE中的二级结构 ,发现胰高血糖素以α螺旋为主要二级结构 ,2 1Ala 胰高血糖素仍有α螺旋结构特征 ,并且含量有所增大 .利用兔升血糖试验 ,发现2 1Ala 胰高血糖素生物活性比重组胰高血糖素减少 51 % (P <0 .0 1 ) .显示天然胰高血糖素第 2 1位氨基酸天冬氨酸与形成α螺旋结构关系不大 ,但在发挥胰高血糖素的生物功能中有重要作用 ,与其可作为钙离子结合位点 ,参与胰高血糖素和受体结合的潜在功能密切相关 .

Changes of Glucagon Structure and Function by ~(21)Ala Site-Directed Mutagenesis

Glucagon is a peptide hormone composed of 29 amino acids.Its biological function is to promote glucogen catabolism,so that it is possible to treat diabetes patients with glucagon antagonist in future.Using recombinant glucagon expression system,the 21st aspartic acid was changed to be alanine by means of site directed mutagenesis.Glucagon gene mutation was testified by DNA sequencing.After expression, 21 Ala glucagon was purified by affinity chromatography and C8 RP HPLC. 21 Ala glucagon was identified by relative molecular mass assay with MS.The comparison between the secondary structures of recombinant glucagon and 21 Ala glucagon by CD indicated that α helix content increased in 21 Ala glucagon.By means of rabbit blood glucose test after glucagon or 21 Ala glucagon treatment,it showed that the biological activity of 21 Ala glucagon was 51% less than that of glucagon ( P <0.01).The results suggest that 21 Asp has no relationship with α helix formation of glucagon secondary structure,but it may be very important in glucagon biological activity.Because 21 Asp may act as a binding site of Ca 2+ ,it will promote the binding of glucagon and glucagon receptor with Ca 2+ participation.