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High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes
Published on: March 24, 2015
Isolation and structural characterization of three isoforms of recombinant consensus alpha interferon
M L Klein1, T D Bartley, J M Davis
1Amgen Inc., Thousand Oaks, California 91320.
Archives of Biochemistry and Biophysics
|February 1, 1990
Summary
Recombinant alpha interferon expressed in E. coli showed three subforms. The N-terminus of one subform was acetylated, indicating post-translational modification in Escherichia coli.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Chemistry
Background:
- Recombinant DNA technology enables the production of therapeutic proteins.
- Alpha interferon is a crucial cytokine with diverse biological functions.
- Understanding protein heterogeneity is vital for drug development.
Purpose of the Study:
- To characterize the heterogeneity of recombinant consensus alpha interferon.
- To investigate the structural basis for observed subforms.
- To identify post-translational modifications in Escherichia coli-expressed interferon.
Main Methods:
- Recombinant protein expression in Escherichia coli.
- Purification using isoelectric focusing and reverse-phase HPLC.
- Automated N-terminal amino acid sequencing.
- Mass spectrometry and amino acid analysis of tryptic peptides.
Main Results:
- Three distinct isoelectric subforms (pI 6.1, 6.0, 5.7) were identified.
- Subforms showed no apparent differences in biological activity or tertiary structure.
- The pI 6.1 and 6.0 subforms corresponded to methionyl and des-methionyl variants.
- The pI 5.7 subform exhibited an N-terminal blockage, identified as acetylation of the N-terminal cysteine residue.
Conclusions:
- Recombinant alpha interferon produced in E. coli exhibits heterogeneity due to N-terminal modifications.
- Acetylation of the N-terminal cysteine is a post-translational modification occurring in E. coli.
- This heterogeneity does not affect biological activity or tertiary structure.
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