Cooper D L, Baptist E W, Enghild J, Lee H, Isola N, Klintworth G K
Department of Pathology, Duke University Medical Center, Durham, NC.
Curr Eye Res. 1990 Aug;9(8):781-6. doi: 10.3109/02713689008999573.
The most abundant soluble protein of bovine cornea, BCP 54 (Bovine Corneal Protein, molecular weight 54 kD) was isolated and digested under both limited and complete digestion conditions with Staphylococcus aureus V8 protease. The fragments resulting from limited digestion were separated by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to a polyvinylidene difluoride membrane, visualized by Coomassie Blue staining, cut out, and submitted to N-terminal protein sequence analysis. Complete digestion fragments were separated on a Vydac C18 reverse-phase HPLC, collected, and concentrated prior to sequencing. Using this method, we obtained amino acid sequence data from three internal V8 protease derived fragments of BCP 54 and a number of HPLC fragments. Comparison of these amino acid sequences, corresponding to 30% of the BCP 54 molecule, to those sequences contained within release 22 of the National Biomedical Research Foundation Protein Identification Resource revealed no extended sequence similarity of known proteins to BCP54.
牛角膜中含量最丰富的可溶性蛋白，即BCP 54（牛角膜蛋白，分子量54kD），在有限消化和完全消化条件下，均用金黄色葡萄球菌V8蛋白酶进行分离和消化。有限消化产生的片段通过一维十二烷基硫酸钠聚丙烯酰胺凝胶电泳进行分离，转移至聚偏二氟乙烯膜上，用考马斯亮蓝染色进行可视化，切下后进行N端蛋白质序列分析。完全消化的片段在Vydac C18反相高效液相色谱上进行分离，收集并浓缩后进行测序。使用该方法，我们获得了BCP 54的三个内部V8蛋白酶衍生片段以及多个高效液相色谱片段的氨基酸序列数据。将这些对应于BCP 54分子30%的氨基酸序列与国家生物医学研究基金会蛋白质鉴定资源库第22版中包含的序列进行比较，结果显示已知蛋白质与BCP54没有广泛的序列相似性。
