[Soluble expression of peptide containing MUC1/Y-specific epitope in Escherichia coli and preparation of the antibody].
MUC1 mucin is a high molecular weight, type I transmembrane glycoprotein. High and aberrant expression of MUC1 is observed in various types of tumors, which make it an ideal target for tumor biotherapy as well as a biomarker for tumor diagnosis and prognosis. MUC1/Y is an isoform of MUC1 generated by alternative splicing. Specific expression of MUC1/Y in breast cancer as well as its involvement in tumor cell signal transduction have been reported. In order to purify peptides containing MUC1/Y-specific epitope in E. coli and prepare MUC1/Y-specific antibody, DNA fragment encoding the MUC1/Y-specific peptide was amplified by PCR using MUC1/Y full length cDNA as the template and cloned into fusion expression vector pGEX-2T, resulting pGEX-Y30. DNA sequencing was performed to confirm the correct amplification and orientation of the target sequence. Competent E. coli DH5alpha was transformed with pGEX-Y30 and the expression was induced for 4-5 hours in 0.2 mmol/L IPTG at 30 degrees C and 37 degrees C. Expressed proteins were released from the cells by ultrasonication or B-PER II reagent treatments. The fusion protein GST-Y30 were purified by affinity and anion exchange columns and identified by SDS-PAGE and Western-blotting. Polyclonal antibody was prepared by immunizing rabbits with the GST-Y30 protein for 4 times with intervals of 3 weeks and purified by GST column. Western blotting, ELISA and immunohistochemistry analysis were carried out using the purified antibody to confirm its MUC1/Y-binding capacity and specificity. The expressed fusion protein GST-Y30 is about 31 kD in size and represented about 20% of total cellular proteins. The majority of the GST-Y30 protein existed as soluble form when the induction was carried out at both 30 degrees C and 37 degrees C. After the two-step purification, the purity of GST-Y30 was about 94%. The titer of polyserum generated by GST-Y30 immunization was 1:320,000 by ELISA. The antiserum showed MUC1/Y specificity and can recognize MUC1/Y on MCF7 cell. The MUC1/Y-specific polyclonal antibody can be used for studying the role of MUC1/Y in carcinogenesis.
Zhang
Li-Xin
LX
Departmert of Tumor Molecular Biology, Bejing North Taiping Hospital and Beyiing Institute of Basic Medical Sciences, Beijing 100039, China. Zhanglx@nic.bmi.ac.cn
Li
Chun-Hai
CH
Sun
Li-Ya
LY
Wang
Miao
M
Lu
Hao-Jun
HJ
D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D000918:Antibody Specificity; D045744:Cell Line, Tumor / Q000378:metabolism; D000939:Epitopes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D008957:Models, Genetic; D018396:Mucin-1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits
D000906:Antibodies; D000939:Epitopes; D018396:Mucin-1; D010455:Peptides