Kozak sequence libraries for systematically characterizing transgenes across expression levels

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Mammalian overexpression systems lack control over protein expression levels and abundance.
• Interpreting how protein sequence and expression affect cellular phenotypes is challenging.
• Systematic modulation of protein abundance is crucial for understanding gene function.

Purpose of the Study:

• To explore the translation initiation sequence (Kozak sequence) for modulating protein steady-state abundance and cellular function.
• To establish a method for linking protein sequence, expression levels, and functional outcomes.
• To identify disease-associated gene variants affecting protein expression.

Main Methods:

• Performed sort-seq on a randomized library of 4042 Kozak sequences preceding the start codon.
• Integrated a single sequence at a shared genomic site in HEK 293T cells for controlled expression.
• Quantified protein steady-state abundances across a range of Kozak sequences.

Main Results:

• Demonstrated a ~100-fold range of protein steady-state abundances by manipulating the Kozak sequence.
• Identified human germline variants with predicted expression-reducing Kozak substitutions in disease genes.
• Showcased modulation of ACE2 cell surface abundance to control SARS-like coronavirus pseudotyped particle infection rates.

Conclusions:

• The Kozak sequence serves as a powerful tool for systematically controlling protein abundance.
• This approach provides a methodological foundation for connecting protein sequence, abundance, and cellular function.
• The findings have implications for understanding disease mechanisms and developing targeted therapies.