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Protein purification is essential in the study of protein structure and function, and the development of novel therapeutics. Many studies require purifying multiple proteins at once, increasing the demand for improved purification methods. We hypothesized that multiple chromatography columns could be interfaced with a multi-well collection plate for rapid and convenient protein purification without the need of expensive instrumentation. As such, we developed a multi-column plate adapter (MCPA), which provides an economical yet versatile and time efficient, high-throughput protein purification system. The MCPA system simultaneously purified milligrams of different proteins under gravity or under vacuum for faster purification. The MCPA handles up to twenty-four 12 mL columns and multiple MCPA's in sequence allow milligram-scale purification of 96 different samples with relative ease. We also used the MCPA system for large scale affinity purification of four proteins, providing sufficient yields and purity for protein crystallization and biophysical characterization. The MCPA system is ideal for optimizing resin type and volume or any other purification parameter by customizing individual columns during the same purification. The high-throughput and versatile nature of this system should prove to be useful in obtaining adequate amounts of protein for subsequent analyses in any laboratory setting.

Chronic hypoxia (CH)-induced pulmonary hypertension (PH) results, in part, from T helper-17 (TH17) cell-mediated perivascular inflammation. However, the antigen(s) involved is unknown. Cellular immunity to collagen type V (col V) develops after ischemia-reperfusion injury during lung transplant and is mediated by naturally occurring (n)TH17 cells. Col5a1 gene codifies for the α1-helix of col V, which is normally hidden from the immune system within type I collagen in the extracellular matrix. COL5A1 promoter analysis revealed nuclear factor of activated T cells, cytoplasmic 3 (NFATc3) binding sites. Therefore, we hypothesized that smooth muscle NFATc3 upregulates col V expression, leading to nTH17 cell-mediated autoimmunity to col V in response to CH, representing an upstream mechanism in PH development. To test our hypothesis, we measured indexes of PH in inducible smooth muscle cell (SMC)-specific NFATc3 knockout (KO) mice exposed to either CH (380 mmHg) or normoxia and compared them with wild-type (WT) mice. KO mice did not develop PH. In addition, COL5A1 was one of the 1,792 genes differentially affected by both CH and SMC NFATc3 in isolated intrapulmonary arteries, which was confirmed by RT-PCR and immunostaining. Cellular immunity to col V was determined using a trans vivo delayed-type hypersensitivity assay (Tv-DTH). Tv-DTH response was evident only when splenocytes were used from control mice exposed to CH but not from KO mice, and mediated by nTH17 cells. Our results suggest that SMC NFATc3 is important for CH-induced PH in adult mice, in part, by regulating the expression of the lung self-antigen COL5A1 protein contributing to col V-reactive nTH17-mediated inflammation and hypertension.

Chronic hypoxia (CH)-induced pulmonary hypertension (PH) is a progressive and often fatal consequence of chronic lung diseases, chronic exposure to high altitude and sleep apnea. PH results from arterial remodeling, enhanced vasoreactivity, and Th17 cell-mediated perivascular inflammation. Naturally occurring “nTH17” cells, can be detected in fetal life and are in homeostatic equilibrium with natural T regulatory cells. nTH17 cells are specific for a limited range of self-antigens, including type V collagen (colV). ColV is normally hidden from the immune system within type I col in the extracellular matrix of the lungs. We have shown that type V collagen is a self-antigen uncovered by CH. On the other hand, we have shown that mice lacking nuclear factor of activated T cells isoform c3 (NFATc3) globally and specifically in smooth muscle (SMC-NFATc3 KO) are protected from CH-induced PH. No differences in cardiac function were detected between SMC-NFATc3 KO and control mice using a Vevo LAZR-X ultrasound system before CH. In silico analysis of Col5A1 promoter shows NFATc3 binding sites. Therefore, we hypothesized that smooth muscle NFATc3 upregulates Col5A1 expression contributing to the development of acute autoreactivity to collagen V in response to CH, representing an upstream mechanism in PH development. Col5A1 was one of the 1792 genes differentially affected by both CH and smooth muscle NFATc3 in isolated pulmonary arteries (RNA Illumina Sequencing). Col5A1 mRNA increased significantly after 5 days of CH in control mice (fold change from normoxia±SD=2.2±0.8, n=5) vs. SMC-NFATc3 KO (0.8±0.3, n=3, p-4 ”] ± SD of naïve recipient mice injected with cells from CH control mice 25.6±4.2, n=8 vs. CH SMC-NFATc3 KO mice 5.3±0.5, n=7; p Our results suggest that smooth muscle NFATc3 is important for CH-induced PH in adult mice, in part, by regulating the expression of the lung self-antigen ColV contributing to pulmonary vascular inflammation.