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Protein aggregation during overexpression limited by peptide extensions with large net negative charge
Yian-Biao Zhang1, Jason Howitt, Sean McCorkle
1Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA.
Protein Expression and Purification
|July 14, 2004
Summary
Adding negatively charged peptides to proteins like CAR D1 can improve their folding during overexpression. This strategy enhances protein solubility and correct folding, offering insights into spontaneous protein folding mechanisms.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Engineering
Background:
- Overexpression of human coxsackie and adenovirus receptor immunoglobulin (Ig) variable-type domain (CAR D1) in Escherichia coli cytoplasm leads to misfolding.
- Fusion to a C-terminal peptide partially rescues CAR D1 folding.
Purpose of the Study:
- Investigate peptide sequence features for solubilization and folding of CAR D1.
- Examine similar Ig variable-type domains from A33 antigen and myelin P-zero proteins.
Main Methods:
- Utilized peptide extensions with varying net negative charges.
- Assessed protein solubilization and folding status.
- Analyzed structurally homologous proteins.
Main Results:
- Peptide extensions with net negative charge > -6 fully solubilized CAR D1, with ~50% correct folding.
- Peptides with net charge -12 partially solubilized A33 and P-zero domains.
- Only the P-zero domain showed correct folding; A33 formed soluble microaggregates.
Conclusions:
- High net negative charge on peptide extensions promotes electrostatic repulsion, reducing protein aggregation.
- This reduction in aggregation allows for spontaneous folding into native conformations.
- Analyzing homologous proteins during overexpression can reveal sequence-dependent folding efficiencies.