N-Terminus Polypeptide Engineering for Stability
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Solution Overview
Problem
Polypeptides expressed intracellularly in recombinant eukaryotic host cells often suffer from reduced stability and biological activity due to co- or post-transcriptional processing such as N-terminal acetylation.
Innovation Solution
Optimization of the amino acid sequence of polypeptides by introducing modifications within the N-terminal region to enhance stability and biological activity, including increased thermodynamic and kinetic stability, and altering the ratio of non-N-terminally acetylated to N-terminally acetylated forms.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If polypeptides are expressed in an intracellular form in recombinant eukaryotic host cells, then downstream processing costs are reduced and polypeptide production is simplified, but polypeptide stability is reduced due to co- or post-transcriptional processing such as N-terminal acetylation
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence at the N-terminal region of polypeptides. Specifically, the invention introduces specific amino acid modifications (such as substituting Serine at position 1 with Alanine or Glycine, or substituting Serine at position 2 with Alanine) to alter the chemical properties of the polypeptide's N-terminus. These sequence parameter changes prevent N-terminal acetylation and other co-translational modifications, thereby maintaining polypeptide stability while preserving the benefits of intracellular expression.
2Productivity
If N-terminal acetylation occurs during intracellular expression, then polypeptide production is efficient, but polypeptide biological activity is reduced
Solution Approach 1:
The patent converts the harmful effect of N-terminal acetylation into a beneficial outcome by strategically introducing amino acid modifications at the N-terminus. These modifications (such as using Alanine or Glycine at position 1 or 2) are specifically chosen to block acetylation sites while maintaining or enhancing polypeptide stability and biological activity. The invention transforms the potential harm of acetylation into a benefit by designing sequences that actively prevent unwanted modifications.
3Stability of the object's composition
If amino acid modifications are introduced at the N-terminal region to increase stability, then polypeptide stability increases, but the complexity of protein engineering increases
Solution Approach 1:
The patent applies local quality by focusing amino acid modifications specifically at the N-terminal region of polypeptides, rather than engineering the entire protein sequence. The invention identifies critical positions (such as position 1 and 2) where specific amino acid substitutions (e.g., Serine to Alanine or Glycine) can be made to achieve stability enhancement. This localized approach minimizes engineering complexity while maximizing the impact on polypeptide stability and resistance to N-terminal acetylation.
Data Source
AI summary
Heterologous polypeptides expressed in a recombinant eukaryotic host cell may exhibit reduced stability and/or biological activity when expressed in an intracellular form. The present disclosure provides a heterologous polypeptide comprising at least one amino acid modifications which increases its stability and/or biological activity when expressed intracellularly in a recombinant eukaryotic host cell.