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1.
Nat Methods ; 15(5): 330-338, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29638227

RESUMO

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. We further validated PCRP mAbs by means of multiple experimental applications, including immunoprecipitation, immunoblotting, chromatin immunoprecipitation followed by sequencing (ChIP-seq), and immunohistochemistry. We also conducted a meta-analysis that identified critical variables that contribute to the generation of high-quality mAbs. All validation data, protocols, and links to PCRP mAb suppliers are available at http://proteincapture.org.


Assuntos
Anticorpos Monoclonais/imunologia , Análise Serial de Proteínas/métodos , Fatores de Transcrição/metabolismo , Animais , Clonagem Molecular , Bases de Dados Factuais , Feminino , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Reprodutibilidade dos Testes
2.
Biochemistry ; 52(7): 1236-48, 2013 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-23346931

RESUMO

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. More importantly, 2D IR correlation spectroscopy provides unique information about the similarities and differences in dynamics between these related proteins. The thermally induced molecular behavior of human centrins can be used to predict biological target interactions that have a relative dependence on calcium affinity. This information is essential for understanding why certain isoforms may be used to rescue a phenotype and therefore also for explaining the different functions these proteins may have in vivo. Furthermore, this comparative approach can be applied to the study of recombinant therapeutic protein candidates for the treatment of disease states.


Assuntos
Proteínas de Ligação ao Cálcio/química , Cálcio/metabolismo , Proteínas de Ciclo Celular/química , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Ligação ao Cálcio/metabolismo , Varredura Diferencial de Calorimetria , Proteínas de Ciclo Celular/metabolismo , Dicroísmo Circular , Humanos , Dados de Sequência Molecular , Conformação Proteica , Desnaturação Proteica , Espectroscopia de Infravermelho com Transformada de Fourier , Análise Espectral/métodos
3.
Vaccines (Basel) ; 11(5)2023 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-37243079

RESUMO

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 6.8, we increased expression levels to 101 mg/L in a 50 L bioreactor, nearly twice the previously reported titer value. A battery of analytical methods was developed in accordance with current good manufacturing practices to ensure a quality biopharmaceutical. Imaged capillary isoelectric focusing verified proper glycosylation of gp145; dynamic light scattering confirmed the trimeric arrangement; and bio-layer interferometry and circular dichroism analysis demonstrated native-like properties (i.e., antibody binding and secondary structure). MALDI-TOF mass spectrometry was used as a multi-attribute platform for accurate mass determination, glycans analysis, and protein identification. Our robust analysis demonstrates that our gp145 product is very similar to a reference standard and emphasizes the importance of accurate characterization of a highly heterogeneous immunogen for the development of an effective vaccine. Finally, we present a novel guanosine microparticle with gp145 encapsulated and displayed on its surface. The unique properties of our gp145 microparticle make it amenable to use in future preclinical and clinical trials.

4.
Artigo em Inglês | MEDLINE | ID: mdl-33571843

RESUMO

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, 3.5 µm) column, fitted with a C8 guard column (Zorbax 300SB-C8, 2.1 × 12.5 mm, 5 µm), using 0.1% TFA and 2% n-propanol in LC-MS water as mobile phase A and 0.1% TFA, 70% isopropanol, and 20% acetonitrile in LC-MS water as mobile phase B. The column temperature was 80 °C, the flow rate was 0.4 mL/min and the absorbance was monitored at 280 nm. The procedures and capabilities of the method were evaluated against the criteria for linearity, limit of detection (LOD), accuracy, repeatability, and robustness as defined by the International Conference on Harmonization (ICH) 2005 Q2(R1) guidelines. Two different variants of the HIV-1 envelope protein (Env), CO6980v0c22 gp145 and SF162 gp140, were analyzed and their retention times were found to be different. The method showed good linearity (R2 = 0.9996), a lower LOD of 2.4 µg/mL, and an average recovery of 101%. The analysis includes measurements of accuracy, inter-user precision, and robustness. Overall, we present a RP-HPLC method that could be applied for the quantitation of cell culture titers for this and other variants of HIV Env following ICH guidelines.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Cromatografia de Fase Reversa/métodos , Produtos do Gene env do Vírus da Imunodeficiência Humana/análise , Animais , Células CHO , Técnicas de Cultura de Células , Cricetinae , Cricetulus , Limite de Detecção , Modelos Lineares , Proteínas Recombinantes/análise , Proteínas Recombinantes/metabolismo , Reprodutibilidade dos Testes , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo
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