Dolby N, Dombrowski K E, Wright S E
Department of Internal Medicine, Texas Tech University Health Sciences Center, 1400 Wallace Boulevard, Amarillo, Texas, 79106, USA.
Protein Expr Purif. 1999 Feb;15(1):146-54. doi: 10.1006/prep.1998.1002.
Adenocarcinomas of glandular tissues produce a hypoglycosylated form of a normal glycoprotein (mucin) that elicits an immune response. A tumor-specific epitope of mucin occurs in a 20-amino-acid, tandemly repeated domain of human MUC1 mucin. A synthetic gene encoding five tandem repeats of the tumor-specific epitope of human mucin (m5tr) was designed for efficient cloning and expression in Escherichia coli for subsequent use in preparing reagent quantities of the mucin 5 tandem repeat (mtr5) polypeptide. The synthetic gene was cloned in the correct reading frame into the maltose-binding protein (MBP) fusion expression vector pMAL-p2. Bacterial clones containing the mucin synthetic gene (m5tr) were shown to produce the intended recombinant fusion protein, MBP-mtr5. The fusion protein represents a significant fraction of the cell protein, 50% or more of which is secreted into the periplasm. The MBP-mtr5 protein is largely intact and easily prepared in sufficient quantity and purity for preliminary structure-function studies.
腺组织腺癌产生一种正常糖蛋白（粘蛋白）的低糖基化形式，可引发免疫反应。粘蛋白的肿瘤特异性表位出现在人MUC1粘蛋白的一个20个氨基酸的串联重复结构域中。设计了一个编码人粘蛋白肿瘤特异性表位五个串联重复序列（m5tr）的合成基因，以便在大肠杆菌中高效克隆和表达，随后用于制备试剂级别的粘蛋白5串联重复序列（mtr5）多肽。该合成基因以正确的阅读框克隆到麦芽糖结合蛋白（MBP）融合表达载体pMAL-p2中。含有粘蛋白合成基因（m5tr）的细菌克隆显示可产生预期的重组融合蛋白MBP-mtr5。融合蛋白占细胞蛋白的很大一部分，其中50%或更多分泌到周质中。MBP-mtr5蛋白基本完整，易于大量制备且纯度足以用于初步的结构功能研究。
