Helmke Steve M, Lu Stephen M, Harmon Michelle, Glasford Joe W, Larsen Tricia D, Kwok Stanley C, Hodges Robert S, Perryman M Benjamin
Deparment of Pediatrics, University of Colorado at Denver and Health Sciences Center at Fitzsimons, Aurora, CO 80045, USA.
J Mol Recognit. 2006 May-Jun;19(3):215-26. doi: 10.1002/jmr.769.
Myotonic dystrophy protein kinase (DMPK) was the initial representative of a ubiquitous protein kinase family that regulates cell size and shape. DMPK is highly expressed in heart and skeletal muscle and transgenic over-expression induces cardiac hypertrophy. The characterization of DMPK has been limited by the paucity of immunological reagents with high affinity and well-defined specificity. Amino acid sequence data was used to predict the surface exposure of the coil-coiled domain of DMPK. These exposed amino acids were substituted into an extremely stable coiled-coil template to produce a peptide antigen. Sera from mice immunized with the peptide conjugated to keyhole limpet hemocyanin were screened against recombinant DMPK using Western blots. Murine spleens expressing DMPK antibodies were used to produce hybridoma cell lines. Hybridoma supernatants were further screened against recombinant DMPK and four clonal hybridoma cell lines expressing DMPK antibodies were generated. These four monoclonal antibodies recognized recombinant DMPK in Western blots of COS-1 cell lysates expressing high levels of recombinant DMPK and immunoprecipitated recombinant DMPK from COS-1 cell lysates. The identity of the immunoprecipitated DMPK was confirmed by MALDI-TOF mass spectrometry and peptide mass fingerprinting. DMPK was the only protein detected in the immunoprecipitates, indicating the high specificity of the antibodies. Western blots immunostained with two of the monoclonal antibodies specifically recognized the two isoforms of endogenous DMPK, DMPK-1 and DMPK-2, that are expressed at low levels in the human heart. The recognition of low amounts of DMPK-1 and DMPK-2 indicates the high affinity of these antibodies. A human heart lysate was subjected to ammonium sulfate precipitation and column chromatography to produce a fraction that was enriched in DMPK. One of the monoclonal antibodies immunoprecipitated endogenous DMPK from this fraction. This antibody was used for immuno-localization studies of an adenoviral DMPK construct, expressed in adult mouse cardiac myocytes. This construct was localized to the intercalated disc, the site of endogenous DMPK, indicating that this antibody is applicable to immuno-localization studies. This study demonstrates the utility of the described procedure for generation of specific monoclonal antibodies with high affinity for epitopes in coiled-coiled domains of mammalian proteins expressed at low levels.
强直性肌营养不良蛋白激酶（DMPK）是一个调节细胞大小和形状的普遍存在的蛋白激酶家族的最初代表。DMPK在心脏和骨骼肌中高度表达，转基因过表达会诱导心脏肥大。DMPK的特性表征受到高亲和力和明确特异性的免疫试剂匮乏的限制。氨基酸序列数据被用于预测DMPK卷曲螺旋结构域的表面暴露情况。这些暴露的氨基酸被替换到一个极其稳定的卷曲螺旋模板中以产生一种肽抗原。用与钥孔血蓝蛋白偶联的肽免疫的小鼠血清通过蛋白质印迹法针对重组DMPK进行筛选。表达DMPK抗体的小鼠脾脏被用于产生杂交瘤细胞系。杂交瘤上清液进一步针对重组DMPK进行筛选，产生了四个表达DMPK抗体的克隆杂交瘤细胞系。这四种单克隆抗体在表达高水平重组DMPK的COS-1细胞裂解物的蛋白质印迹中识别重组DMPK，并从COS-1细胞裂解物中免疫沉淀重组DMPK。通过基质辅助激光解吸电离飞行时间质谱（MALDI-TOF）和肽质量指纹图谱确认了免疫沉淀的DMPK的身份。DMPK是免疫沉淀中检测到的唯一蛋白质，表明抗体具有高特异性。用其中两种单克隆抗体进行免疫染色的蛋白质印迹特异性识别内源性DMPK的两种异构体DMPK-1和DMPK-2，它们在人类心脏中低水平表达。对低含量的DMPK-1和DMPK-2的识别表明这些抗体具有高亲和力。对人心脏裂解物进行硫酸铵沉淀和柱色谱以产生富含DMPK的级分。其中一种单克隆抗体从该级分中免疫沉淀内源性DMPK。该抗体用于对在成年小鼠心肌细胞中表达的腺病毒DMPK构建体进行免疫定位研究。该构建体定位于闰盘，即内源性DMPK的位点，表明该抗体适用于免疫定位研究。这项研究证明了所描述的程序对于产生对低水平表达的哺乳动物蛋白卷曲螺旋结构域中的表位具有高亲和力的特异性单克隆抗体的实用性。
