Optimizing the Production of gp145, an HIV-1 Envelope Glycoprotein Vaccine Candidate and Its Encapsulation in Guanosine Microparticles.

We have developed a pipeline to express, purify, and characterize HIV envelope protein (Env) gp145 from Chinese hamster ovary cells, to accelerate the production of a promising vaccine candidate. First in shake flasks, then in bioreactors, we optimized the growth conditions. By adjusting the pH to 6.

A reversed phase HPLC method for the quantification of HIV gp145 glycoprotein levels from cell culture supernatants.

A reversed phase high performance liquid chromatography (RP-HPLC) method was developed for the quantitative determination of recombinant HIV-1 gp145 produced in CHO-K1 cells, as measured directly from culture supernatants. Samples were diluted in 50% D-PBS and 50% PowerCHO-2 (PC2) spent medium, and resolved on a Zorbax 300SB-C8 Rapid Resolution (2.1 × 50 mm, 3.

A toolbox of immunoprecipitation-grade monoclonal antibodies to human transcription factors.

A key component of efforts to address the reproducibility crisis in biomedical research is the development of rigorously validated and renewable protein-affinity reagents. As part of the US National Institutes of Health (NIH) Protein Capture Reagents Program (PCRP), we have generated a collection of 1,406 highly validated immunoprecipitation- and/or immunoblotting-grade mouse monoclonal antibodies (mAbs) to 737 human transcription factors, using an integrated production and validation pipeline. We used HuProt human protein microarrays as a primary validation tool to identify mAbs with high specificity for their cognate targets.

Relative stability of human centrins and its relationship to calcium binding.

Centrins are calcium binding proteins that belong to the EF-hand superfamily with diverse biological functions. Herein we present the first systematic study that establishes the relative stability of related centrins via complementary biophysical techniques. Our results define the stepwise molecular behavior of human centrins by two-dimensional infrared (2D IR) correlation spectroscopy, the change in heat capacity and enthalpy of denaturation by differential scanning calorimetry, and the relative stability of the helical regions of centrins by circular dichroism.