Prokaryotic Expression,Purification,Antibody Production of a NH_2-Terminal Fragment of mCLCA3 Protein and Analysis of mClCA3 Protein Expression in Asthmatic Mouse Lung

mCLCA3 is a member of calcium activated chloride channel（CACC） family that may play an important role in mucin packaging and secretion in asthmatic and cystic fibrosis lung. To study the protein structure and expression of mCLCA3 in asthmatic mouse lung, an N-terminal 269 amino acid peptide of mCLCA3 was expressed in E. coli, purified to homogeneity and rabbit polyclonal antibodies against this peptide were generated. Immunohistochemistry of asthmatic mouse lung using the antibody indicated exclusive mCLCA3 expression in mucin granules of goblet cells in airway surface and lumen, Immunoblot analysis of lavage fluid from asthmatic mouse lung revealed a single 90 kDa protein form of mClCA3. The results demonstrate that the 90 kDa N-terminal peptide, neither the flail-length protein nor the reported N-terminal 35 kDa cleaved form of mClCA3 is the major functional form involved in the packaging and exocytosis of mucin granules in asthmatic goblet cells.

Antibody Production for a Rapid Fluorescence Polarization Immunoassay of Estrone

Estrone has been identified as a potential endocrine-disrupting chemical （EDC）[1]. Estrone is usually quantified by gas chromatography-mass spectrometry （GC-MS）, GC-MS/MS, high performance liquid chromatography （HPLC）, HPLC- MS, and HPLC-MS/MS, etc.[2-3]. Meanwhile, several immunoassays based on radioimmunoassay, enzyme linked immunosorbent assay （ELISA） or chemiluminescence immunoassay （CLIA） for determination of estrone in real samples have been developed[2＇4]. Although these methods are sensitive, they need multistage separation and are thus time-consuming and laborious. A very promising way for the simplification of immunoassays for routine applications is a shift from heterogeneous methods （with separation） to homogeneous assays （without separation）[5]. Fluorescence polarization immunoassay （FPIA） is one of the homogeneous techniques that meets the requirements of a simple, reliable, fast, and cost-effective analysis[6]. Therefore, the present study is focused on the development of FPIA in order to analyze estrone based on antibody production.

HLA antigens and anti-sperm antibody production in Iranian vasectomized men

Dear Editor： Anti-sperm antibodies （ASAs） are composed of numerous antibodies interacting with multiple sperm antigens that play a role in fertility. In males, ASAs cause ＇immune infertility＇ by decreasing sperm counts and normal forms, as well as reducing sperm motility and viability, markedly reducing the likelihood of natural conception. The development of ASA in the male depends on the release of sequestered antigens on germ cells following the disruption of the blood-testis barrier.

Activity and Tissue Expression of Tyrosine Phosphatase PTP-MEG2

Protein tyrosine phosphatases（PTPs） are crucial regulators of signal transduction. Among them, PTP-MEG2 is an intracellular enzyme of 593 amino acid residues with a putative lipid-binding domain at the N-terminus. In the present study, we cloned the full-length form of the enzyme and expressed it in E. coli cells as a 6xHis-tagged protein. The majority of the expressed enzyme was found in the inclusion body of E. coli cell extracts. Upon extraction with a buffer containing urea, the recombinant enzyme was purified to near homogeneity on a single Ni-NTA-agarose column. This procedure resulted in the production of over 100 mg of purified recombinant PTP-MEG2 from 1 L E. coli cell culture. The purified protein displayed a single polypeptide band with expected molecular size on SDS-polyacrylamide gel electrophoresis under reducing conditions. Isolated under denatured conditions in urea, the purified enzyme was re-natured by dialyzing against a refolding buffer. The re-natured enzyme effectively dephosphorylated the common PTP substrate para-nitrophenylphosphate with a specific activity of 2000 units/mg. Meanwhile, the denatured enzyme was used to immunize a rabbit to produce antibodies. The resulting anti- serum had extremely high sensitivity and specificity. When used for Western blot analysis, the anti-serum revealed a wide expression of PTP-MEG2 in many tissues of mice. Together, we developed a highly effective way to purify a large amount of PTP-MEG2 and generated highly sensitive antibodies that can specifically detect endogenous expression of the enzyme in tissues.

Expression of Difficult-to-Express Proteins, Human IL-12 and IFN-<i>γ</i>

It is known to be that lactic acid bacteria induce the IL-12. IL-12 activates NK cells and promotes the production of IFN-γ. IFN-γ activates macrophages, resulting in enhanced phagocytosis and bactericidal activity. We have been investigating fermented foods that activate the immune function. For that purpose, a specific antibody is required. We tried to express IL-12p35 by the usual method, but IL-12p35 was not expressed at all. In the present study, we constructed, purified human IL-12p35 and obtained a specific antibody against IL-12p35. We also purified human IFN-γ and obtained specific antibody against IFN-γ. We have established a method for expressing poorly expressed proteins. The method we have established can be applied to the purification of poorly expressed proteins and antibody production.