Kozak sequence libraries for characterizing transgenes across expression levels

Typical mammalian overexpression systems test protein sequence variants with little control over expression levels and steady-state protein abundances, hindering interpretations of how protein sequence and expression converge to yield phenotypic outcomes. We explored the translation initiation sequence, commonly referred to as the Kozak sequence, as a means to modulate protein steady-state abundance and cellular function. We performed sort-seq on a randomized library of the 6 nucleotides preceding the start codon, amounting to 4,042 sequences. Calibrating the scores revealed a ~100-fold range of protein steady-state abundances possible through manipulation of the Kozak sequence. We identified human germline variants with predicted expression-reducing Kozak substitutions in disease-associated genes. Modulating the cell surface abundance of the host cell receptor ACE2 controlled the rate at which those cells became infected by SARS-like coronavirus spike pseudotyped particles. We demonstrated the potential of the approach by simultaneously testing Kozak libraries with a small panel of coding variants for ACE2 and STIM1. This approach lays the methodological groundwork for linking the causal relationships between protein sequence, abundance, and functional outcome. Overall design: The miRFP670 Kozak library was recombined and sorted in four separate instances to create the four replicates analyzed in our dataset. For the first biological replicate, 120,000 HEK 293T cells were plated per well in a 24-well plate, and each well was transfected with a mixture of 2 µg ACE2 Kozak library plasmid and 8 µL Fugene transfection reagent (Promega, E2692), diluted in 60 µL Opti-MEM (Gibco, 31985070). Eight wells were transfected per attempt and subsequently pooled. Flow cytometry analysis prior to selection showed 2.5% mCherry-positive cells, suggesting that approximately 24,000 cells were independently recombined, corresponding to an estimated 6 independent events per Kozak sequence variant. Following positive and negative selection, sequencing revealed that some key Kozak sequences necessary for calibrating our results with our panel of individual variants were missing. To address the coverage limitations observed in the first replicate, a modified approach was taken for the second replicate. For this replicate, 1 million cells were seeded in a 6-well plate one day before transfection. Cells were transfected with a mixture containing 4 µg ACE2 Kozak library plasmid, 30 ng G790A plasmid, and 1.2 µL Xfect reagent, following the manufacturer's instructions. After positive and negative selection, approximately 8,500 cells from each of the following clonal plasmid-expressing cell lines, G1100A [TTCATCATG], G1101F [GACGACATG], G1102A [CGTCCAATG], G1103A [GCGCGCATG], G1104B [TGGTCAATG], and G1106A [TTGCACATG], were mixed with 10 million library-expressing cells to achieve a diverse range of protein abundance. For the third biological replicate, 1 million cells were seeded in a 6-well plate one day before transfection. Cells were transfected with a mixture containing 4 µg ACE2 Kozak library plasmid, 30 ng G790A plasmid and 30 ng of each Kozak sequence plasmids (G1100A, G1101F, G1102A, G1103A, G1104B, and G1106A), and 1.2 µL Xfect reagent, following the manufacturer's protocol. Following recombination, cells were subjected to positive and negative selection as described before. This approach was designed to enhance the diversity of protein abundance linked to Kozak sequence variations. For both of these replicates, ~ 170,000 cells were collected during each 4-way sort and the cells were cultured for ~48hrs before their genomic DNAs were extracted. The fourth recombination experiment used an independent miRFP670 Kozak library, where miRFP670 was C-terminally fused to the hygromycin resistance gene. Here, cells were transfected in two separate wells of a 6-well plate using Xfect reagent. Each well containing 1 million cells was transfected with 4 µg of Hygromycin Kozak library plasmid mixed with 1.2 µL Xfect reagent.Following recombination, cells underwent positive and negative selection as mentioned above. Roughly 130,000 cells were collected in each bin during 4 way sorting and were cultured for ~48hrs before collecting them for gDNA isolation.