Thioredoxin 1 is responsible for antibody disulfide reduction in CHO cell culture.

During large-scale manufacturing of an IgG1 monoclonal antibody in Chinese hamster ovary (CHO) cells, reduction of the antibody's disulfide bonds was observed. We present evidence that mammalian thioredoxin 1 (TXN1) is the terminal enzyme responsible for this reduction event. We demonstrate a marked prevention of IgG1 disulfide bond reduction in a cell-density dependent manner by knocking down expression of TXN1 via lentivirus transduction of short hairpin RNA.

Detection of ERalpha-SRC-1 interactions using bioluminescent resonance energy transfer.

Bioluminescent Resonance Energy Transfer is a naturally occurring phenomenon that can be exploited to explore protein-protein interactions in real-time in intact cells and cellular extracts. It detects energy transferred between a bioluminescent donor enzyme (Renilla luciferase) fusion protein and a fluorescent (GFP(2), a mutant of Green Fluorescent Protein) acceptor fusion protein. Optimal detection of BRET(2) energy transfer relies on the distance and orientation generated by the fusion proteins. This chapter describes in detail the BRET(2) assay as it is used to examine the physical interaction between the nuclear receptor ERalpha and the transcriptional coactivator SRC-1. Description of methods include selection of donor and acceptor combinations, fusion construct generation and validation, cell culture and transfection, individual fluorescence and luminescence detection, BRET(2) detection, and data analysis.

Measuring ligand-dependent and ligand-independent interactions between nuclear receptors and associated proteins using Bioluminescence Resonance Energy Transfer (BRET).

Bioluminescent resonance energy transfer (BRET2) is a recently developed technology for the measurement of protein-protein interactions in a live, cell-based system. BRET2 is characterized by the efficient transfer of excited energy between a bioluminescent donor molecule (Renilla luciferase) and a fluorescent acceptor molecule (a mutant of Green Fluorescent Protein (GFP2). The BRET2 assay offers advantages over fluorescence resonance energy transfer (FRET) because it does not require an external light source thereby eliminating problems of photobleaching and autoflourescence. The absence of contamination by light results in low background that permits detection of very small changes in the BRET2 signal. BRET2 is dependent on the orientation and distance between two fusion proteins and therefore requires extensive preliminary standardization experiments to conclude a positive BRET2 signal independent of variations in protein titrations and arrangement in tertiary structures. Estrogen receptor (ER) signaling is modulated by steroid receptor coactivator 1 (SRC-1). To establish BRET2 in a ligand inducible system we used SRC-1 as the donor moiety and ER as the acceptor moiety. Expression and functionality of the fusion proteins were assessed by transient transfection in HEK-293 cells followed by Western blot analysis and measurement of ER-dependent reporter gene activity. These preliminary determinations are required prior to measuring nuclear receptor protein-protein interactions by BRET2. This article describes in detail the BRET2 methodology for measuring interaction between full-length ER and coregulator proteins in real-time, in an in vivo environment.

Expression of estrogen receptor coregulators in normal and malignant human endometrium.

OBJECTIVES: To compare the expression of nuclear receptor coregulators in normal and malignant human endometrium and to identify any relationship to grade, stage, age, depth of myometrial invasion, estrogen receptor alpha (ERalpha), or progesterone receptor (PR) expression. METHODS: Gene expression of SRC-1, SRC-2, SRC-3, N-CoR, SMRT, ERalpha, and PR was measured in 26 samples of normal endometrium and 30 primary endometrial carcinomas using real-time RT-PCR. ERalpha protein expression of each tissue was also measured by Western blot. RESULTS: . All coregulators showed significantly increased mRNA expression in endometrial carcinoma as compared to normal endometrium. The mRNA expression of each coregulator showed a high correlation with ERalpha mRNA, PR mRNA, and with the other coregulators in both normal and malignant endometrium. In the normal endometrium, SRC-1 mRNA expression was positively correlated with ERalpha protein expression and SRC-3 mRNA expression was positively correlated with patient age. No relationship was found between coregulator mRNA expression and grade, stage, or depth of myometrial invasion. CONCLUSION: The nuclear receptor coregulators SRC-1, SRC-2, SRC-3, N-CoR, and SMRT were found to be up-regulated in malignant endometrium. Our findings suggest that these proteins may have a role in the development of endometrial carcinoma.