Free M L, Gordon R B, Keough D T, Beacham I R, Emmerson B T, de Jersey J
Department of Biochemistry, University of Queensland, Australia.
Biochim Biophys Acta. 1990 Oct 23;1087(2):205-11. doi: 10.1016/0167-4781(90)90206-h.
A plasmid, pRG1, has been constructed by incorporating the coding sequence of human hypoxanthine-guanine phosphoribosyltransferase (HPRT) into the expression vector pT7-7. Expression of human HPRT has been achieved in HPRT- Escherichia coli cells transformed with pRG1 and pGP1-2, as shown by: (1) exclusive labelling with [35S]methionine of a polypeptide with the same mobility as purified human HPRT on SDS-PAGE; and (2) measurement of HPRT activity after cell lysis. Although the majority of the recombinant HPRT was present in the particulate fraction after cell lysis and centrifugation, sufficient HPRT activity was present in the supernatant fraction to allow comparison with the HPRT purified from human erythrocytes and the activity in human haemolysates and lymphoblast lysates. Small differences in electrophoretic mobility on native gels were found between HPRT activity from these sources. The Km values of recombinant HPRT for the substrates 5-phospho-alpha-D-ribosyl-1-pyrophosphate and guanine were compared with those of lymphoblast and erythrocyte HPRT.
通过将人次黄嘌呤 - 鸟嘌呤磷酸核糖转移酶（HPRT）的编码序列整合到表达载体pT7 - 7中构建了一种质粒pRG1。用pRG1和pGP1 - 2转化HPRT⁻大肠杆菌细胞后，已实现人HPRT的表达，如下所示：（1）在SDS - PAGE上，[³⁵S]甲硫氨酸对与纯化的人HPRT迁移率相同的多肽进行特异性标记；（2）细胞裂解后测量HPRT活性。虽然细胞裂解和离心后大部分重组HPRT存在于颗粒部分，但上清液部分存在足够的HPRT活性，以便与从人红细胞中纯化的HPRT以及人溶血产物和淋巴母细胞裂解物中的活性进行比较。发现这些来源的HPRT活性在天然凝胶上的电泳迁移率存在微小差异。将重组HPRT对底物5 - 磷酸 - α - D - 核糖基 - 1 - 焦磷酸和鸟嘌呤的Km值与淋巴母细胞和红细胞HPRT的Km值进行了比较。
