Munson R, Tolan R W
Edward Mallinckrodt Department of Pediatrics, Washington University, School of Medicine, St. Louis, Missouri.
Infect Immun. 1989 Jan;57(1):88-94. doi: 10.1128/iai.57.1.88-94.1989.
The structural gene for the porin of Haemophilus influenzae type b, designated outer membrane protein P2, was cloned, and the DNA sequence was determined. An oligonucleotide probe generated by reverse translation of N-terminal amino acid sequence data from the purified protein was used to screen genomic DNA. The probe detected a single EcoRI fragment of approximately 1,700 base pairs which was cloned to lambda gt11 and then into M13 and partially sequenced. The derived amino acid sequence indicated that we had cloned the N-terminal portion of the P2 gene. An overlapping approximately 1,600-base-pair PvuII genomic fragment was cloned into M13, and the sequence of the remainder of the P2 gene was determined. The gene for P2 was then reconstructed under the control of the T7 promoter and expressed in Escherichia coli. The N-terminal sequence of the purified protein corresponds to residues 21 through 34 of the derived amino acid sequence. Thus, the protein is synthesized with a 20-amino-acid leader peptide. The Mr of the processed protein is 37,782, in good agreement with the estimate of 37,000 from sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
b型流感嗜血杆菌孔蛋白的结构基因（命名为外膜蛋白P2）被克隆，并测定了其DNA序列。利用根据纯化蛋白N端氨基酸序列数据反向翻译生成的寡核苷酸探针筛选基因组DNA。该探针检测到一个约1700个碱基对的单一EcoRI片段，将其克隆到λgt11中，然后克隆到M13中并进行部分测序。推导的氨基酸序列表明我们克隆到了P2基因的N端部分。将一个约1600个碱基对的重叠PvuII基因组片段克隆到M13中，测定了P2基因其余部分的序列。然后在T7启动子的控制下重建P2基因，并在大肠杆菌中表达。纯化蛋白的N端序列对应于推导氨基酸序列的第21至34位残基。因此，该蛋白是由一个20个氨基酸的前导肽合成的。加工后蛋白的Mr为37782，与十二烷基硫酸钠-聚丙烯酰胺凝胶电泳估计的37000非常一致。
