Use of monobromobimane to resolve two recombinant proteins by reversed-phase high-performance liquid chromatography based on their cysteine content
| Autor: | Shigeko Yamazaki, Donald O. O'Keefe, Ann L. Lee |
|---|---|
| Rok vydání: | 1992 |
| Předmět: |
Chromatography
Lysis Chemistry Organic Chemistry General Medicine Reversed-phase chromatography medicine.disease_cause Biochemistry High-performance liquid chromatography Fusion protein Dithiothreitol Analytical Chemistry law.invention chemistry.chemical_compound law medicine Recombinant DNA Escherichia coli Cysteine |
| Zdroj: | Journal of Chromatography A. 627:137-143 |
| ISSN: | 0021-9673 |
| DOI: | 10.1016/0021-9673(92)87193-c |
| Popis: | A rapid reversed-phase HPLC assay useful for fermentation and downstream process development was developed for monitoring transforming growth factor-α- Pseudomonas aeruginosa exotoxin A 40 (TGFα-PE40). This protein is a chimeric recombinant protein synthesized in Escherichia coli . In the fermentation, full-length TGFα-PE40 is presented along with PE40, an M r ≈ 40 000 C-terminal fragment of TGFα-PE40, which co-purifies with TGFα-PE40 in many cases. A highly efficient reversed-phase HPLC assay using ultraviolet absorbance detection provided excellent resolution of the chimeric protein from the host-cell proteins in the crude cell lysate. However, this technique failed to resolve TGFα-PE40 from PE40, thereby limiting its use for in-process quantitation of the product. In order to resolve these two proteins, we have developed a new technique based on the sulfhydryl specificity of the fluorescent probe monobromobimane. Treatment of in-process samples with dithiothreitol followed by monobromobimane produces fluorescently-labeled TGFα-PE40, but does not label PE40 due to the lack of cysteine residues in this fragment. Thus, reversed-phase HPLC analysis using fluorescence detection provides the selectivity necessary to discriminate between TGFα-PE40 and PE40. |
| Databáze: | OpenAIRE |
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