Schultz D E, Spratt B G, Nicholas R A
Department of Pharmacology, University of North Carolina, Chapel Hill 27599-7365.
Protein Expr Purif. 1991 Oct-Dec;2(5-6):339-49. doi: 10.1016/1046-5928(91)90092-w.
Resistance to penicillin in non-beta-lactamase-producing strains of Neisseria gonorrhoeae (CMRNG strains) is mediated in part by the production of altered forms of penicillin-binding protein 2 (PBP 2) that have a decreased affinity for penicillin. The reduction in the affinity of PBP 2 is largely due to the insertion of an aspartic acid residue (Asp-345a) into the amino acid sequence of PBP 2. Truncated forms of N. gonorrhoeae PBP 2, which differed only by the insertion of Asp-345a, were constructed by placing the region of the penA genes encoding the periplasmic domain of PBP 2 (amino acids 42-581) into an ATG expression vector. When the recombinant PBP 2 molecules were overexpressed in Escherichia coli, insoluble PBP 2 inclusion bodies, which could be isolated by low-speed centrifugation of cell lysates, were formed. These insoluble aggregates were solubilized and the truncated PBP 2 polypeptides were partially purified by cation-exchange chromatography and gel filtration in the presence of denaturant prior to the refolding of the enzyme in vitro. After renaturation, gel filtration was used to separate monomeric soluble PBP 2 from improperly folded protein aggregates and other protein contaminants. A 4-liter culture of induced E. coli cells yielded 1.4 mg of soluble PBP 2 or PBP 2' (PBP 2 containing the Asp-345a insertion), both of which were estimated to be 99% pure. The affinity of soluble PBP 2' for [3H]penicillin G was decreased fourfold relative to that of soluble PBP 2, and their affinities were found to be identical to the affinities of the full-length PBP 2 enzymes that were previously determined in N. gonorrhoeae membranes. Furthermore, soluble PBP 2 displayed a rank order of affinity for several other beta-lactam antibiotics that was consistent with the rank order of affinities previously reported for the native molecules. On the basis of these results, both of these soluble PBPs should be suitable for crystallization and X-ray crystallographic analysis.
在不产生β-内酰胺酶的淋病奈瑟菌菌株（CMRNG菌株）中，对青霉素的耐药性部分是由青霉素结合蛋白2（PBP 2）的改变形式的产生介导的，这些改变形式对青霉素的亲和力降低。PBP 2亲和力的降低主要是由于在PBP 2的氨基酸序列中插入了一个天冬氨酸残基（Asp-345a）。通过将编码PBP 2周质结构域（氨基酸42 - 581）的penA基因区域置于ATG表达载体中，构建了仅因插入Asp-345a而不同的淋病奈瑟菌PBP 2截短形式。当重组PBP 2分子在大肠杆菌中过表达时，形成了不溶性PBP 2包涵体，可通过对细胞裂解物进行低速离心分离得到。这些不溶性聚集体被溶解，截短的PBP 2多肽在体外酶复性之前，通过阳离子交换色谱和在变性剂存在下的凝胶过滤进行部分纯化。复性后，使用凝胶过滤将单体可溶性PBP 2与折叠不当的蛋白质聚集体和其他蛋白质污染物分离。4升诱导的大肠杆菌细胞培养物产生了1.4毫克可溶性PBP 2或PBP 2'（含有Asp-345a插入的PBP 2），两者估计纯度均为99%。可溶性PBP 2'对[3H]青霉素G的亲和力相对于可溶性PBP 2降低了四倍，并且发现它们的亲和力与先前在淋病奈瑟菌膜中测定的全长PBP 2酶的亲和力相同。此外，可溶性PBP 2对其他几种β-内酰胺抗生素的亲和力排序与先前报道的天然分子的亲和力排序一致。基于这些结果，这两种可溶性PBP都应该适合用于结晶和X射线晶体学分析。
