Selection of aptamers for a protein target in cell lysate and their application to protein purification

Functional genomics requires structural and functional studies of a large number of proteins. While the production of proteins through over-expression in cultured cells is a relatively routine procedure, the subsequent protein purification from the cell lysate often represents a significant challenge. The most direct way of protein purification from a cell lysate is affinity purification using an affinity probe to the target protein. It is extremely difficult to develop antibodies, classical affinity probes, for a protein in the cell lysate; their development requires a pure protein. Thus, isolating the protein from the cell lysate requires antibodies, while developing antibodies requires a pure protein. Here we resolve this loop problem. We introduce AptaPIC, Aptamer-facilitated Protein Isolation from Cells, a technology that integrates (i) the development of aptamers for a protein in cell lysate and (ii) the utilization of the developed aptamers for protein isolation from the cell lysate. Using MutS protein as a target, we demonstrate that this technology is applicable to the target protein being at an expression level as low as 0.8% of the total protein in the lysate. AptaPIC has the potential to considerably speed up the purification of proteins and, thus, accelerate their structural and functional studies

Preclinical evaluation of a TEX101 protein ELISA test for the differential diagnosis of male infertility

BACKGROUND: TEX101 is a cell membrane protein exclusively expressed by testicular germ cells and shed into seminal plasma. We previously verified human TEX101 as a biomarker for the differential diagnosis of azoospermia, and developed a first-of-its-kind TEX101 ELISA. To demonstrate the clinical utility of TEX101, in this work we aimed at evaluating ELISA performance in a large population of fertile, subfertile, and infertile men. METHODS: Mass spectrometry, size-exclusion chromatography, ultracentrifugation, and immunohistochemistry were used to characterize TEX101 protein as an analyte in seminal plasma. Using the optimized protocol for seminal plasma pretreatment, TEX101 was measured by ELISA in 805 seminal plasma samples. RESULTS: We demonstrated that TEX101 was present in seminal plasma mostly in a free soluble form and that its small fraction was associated with seminal microvesicles. TEX101 median values were estimated in healthy, fertile pre-vasectomy men (5436 ng/mL, N = 64) and in patients with unexplained infertility (4967 ng/mL, N = 277), oligospermia (450 ng/mL, N = 270), and azoospermia (0.5 ng/mL, N = 137). Fertile post-vasectomy men (N = 57) and patients with Sertoli cell-only syndrome (N = 13) and obstructive azoospermia (N = 36) had undetectable levels of TEX101 (≤0.5 ng/mL). A cut-off value of 0.9 ng/mL provided 100% sensitivity at 100% specificity for distinguishing pre- and post-vasectomy men. The combination of a concentration of TEX101 > 0.9 ng/mL and epididymis-specific protein ECM1 > 2.3 μg/mL provided 81% sensitivity at 100% specificity for differentiating between non-obstructive and obstructive azoospermia, thus eliminating the majority of diagnostic testicular biopsies. In addition, a cut-off value of ≥0.6 ng/mL provided 73% sensitivity at 64% specificity for predicting sperm or spermatid retrieval in patients with non-obstructive azoospermia. CONCLUSIONS: We demonstrated the clinical utility of TEX101 ELISA as a test to evaluate vasectomy success, to stratify azoospermia forms, and to better select patients for sperm retrieval. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12916-017-0817-5) contains supplementary material, which is available to authorized users

Aptamer-based radiopharmaceuticals for diagnostic imaging and targeted radiotherapy of epithelial tumors

In the continuous search for earlier diagnosis and improved therapeutic modalities against cancer, based on our constantly increasing knowledge of cancer biology, aptamers hold the promise to expand on current antibody success, but overcoming some of the problems faced with antibodies as therapeutic or delivery agents in cancer. However, as the first aptamer reached the market as an inhibitor against angiogenesis for the treatment of macular degeneration, aptamers have found only limited applications or interest in oncology, and even less as radiopharmaceuticals for diagnostic imaging and targeted radiotherapy of tumours. Yet, the chemistry for the labelling of aptamers and the options to alter their pharmacokinetic properties, to make them suitable for use as radiopharmaceuticals is now available and recent advances in their development can demonstrate that these molecules would make them ideal delivery vehicles for the development of targeted radiopharmaceuticals that could deliver their radiation load with accuracy to the tumour site, offering improved therapeutic properties and reduced side effects