Lin X L, Wong R N, Tang J
Laboratory of Protein Studies, Oklahoma Medical Research Foundation, Oklahoma City.
J Biol Chem. 1989 Mar 15;264(8):4482-9.
In order to carry out studies on structure and function relationships of porcine pepsinogen using site-directed mutagenesis approaches, the cDNA of this zymogen was cloned, sequenced, expressed in Escherichia coli, and the protein refolded, and purified to homogeneity. Porcine pepsinogen cDNA, obtained from a lambda gt10 cDNA library of porcine stomach contains 1364 base pairs. It contains leader, pro, and pepsin regions of 14, 44, and 326 residues, respectively. In addition, it also contains 5'- and 3'-untranslated regions. Four differences are present between the sequence deduced from the cDNA and the pepsinogen sequence determined previously by protein chemistry methods. Residues P19 (in the pro region) and 263 are asparagines in the cDNA sequence instead of aspartic acids. Isoleucine 230 is not present in the cDNA sequence and residue 242 is a tyrosine in the cDNA instead of an aspartic acid. Porcine pepsinogen cDNA was placed under the control of a tac promoter in a plasmid and expressed in E. coli. The synthesis of pepsinogen was optimized to about 50 mg/liter of culture. The recombinant (r-) pepsinogen, which was insoluble, was recovered by centrifugation, washed, dissolved in 6 M urea in Tris-HCl, pH 8, and refolded by rapid dilution. r-pepsinogen was purified to homogeneity after chromatography on Sephacryl S-300 and fast protein liquid chromatography on a monoQ column. r-pepsinogen contains an additional methionine residue at the NH2 terminus as compared to native (n-) pepsinogen. However, r- and n-pepsinogens are indistinguishable in their intramolecular activation constants. After activation, r- and n-pepsins have the same NH2-terminal sequences as well as Km values. Based on these data, r-pepsinogen was judged suitable for mutagenesis studies. A mutant pepsinogen (D32A) with the active site aspartic acid changed to an alanine was produced and purified. D32A-pepsinogen did not convert to pepsin in acid solution but it bound to pepstatin with an apparent KD of about 5 x 10(-10) M. D32A-pepsinogen possesses no detectable proteolytic activity. These results indicate that (i) intramolecular pepsinogen activation is accomplished by the pepsin active site, and (ii) unlike subtilisin (Carter, P., and Wells, J. A. (1988) Nature 332, 564-568), the active site mutant of pepsin is not enzymically active.
为了利用定点诱变方法开展猪胃蛋白酶原结构与功能关系的研究，克隆了该酶原的cDNA，进行测序，在大肠杆菌中表达，对蛋白质进行重折叠，并纯化至同质。从猪胃的λgt10 cDNA文库获得的猪胃蛋白酶原cDNA包含1364个碱基对。它分别包含14、44和326个残基的前导区、原区和胃蛋白酶区。此外，它还包含5'-和3'-非翻译区。从cDNA推导的序列与先前通过蛋白质化学方法确定的胃蛋白酶原序列之间存在四个差异。原区的第19位残基（P19）和第263位残基在cDNA序列中是天冬酰胺而不是天冬氨酸。第230位异亮氨酸不在cDNA序列中，第242位残基在cDNA中是酪氨酸而不是天冬氨酸。将猪胃蛋白酶原cDNA置于质粒中的tac启动子控制下并在大肠杆菌中表达。胃蛋白酶原的合成优化至约50mg/升培养物。通过离心回收不溶性的重组（r-）胃蛋白酶原，洗涤，溶解于pH8的Tris-HCl中的6M尿素中，并通过快速稀释进行重折叠。在Sephacryl S-300上进行色谱分离和在monoQ柱上进行快速蛋白质液相色谱后，将r-胃蛋白酶原纯化至同质。与天然（n-）胃蛋白酶原相比，r-胃蛋白酶原在NH2末端含有一个额外的甲硫氨酸残基。然而，r-和n-胃蛋白酶原在其分子内活化常数方面无法区分。活化后，r-和n-胃蛋白酶具有相同的NH2末端序列以及Km值。基于这些数据，判断r-胃蛋白酶原适合进行诱变研究。产生并纯化了一种活性位点天冬氨酸变为丙氨酸的突变胃蛋白酶原（D32A）。D32A-胃蛋白酶原在酸性溶液中不会转化为胃蛋白酶，但它以约5×10（-10）M的表观KD与胃蛋白酶抑制剂结合。D32A-胃蛋白酶原不具有可检测的蛋白水解活性。这些结果表明：（i）分子内胃蛋白酶原活化是由胃蛋白酶活性位点完成的；（ii）与枯草杆菌蛋白酶不同（Carter，P.和Wells，J.A.（1988）Nature 332，564-568），胃蛋白酶的活性位点突变体没有酶活性。
