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Structure and function studies of membrane proteins, particularly G protein-coupled receptors and multipass transmembrane proteins, require detergents. We have devised a simple tool, the QTY code (glutamine, threonine, and tyrosine), for designing hydrophobic domains to become water soluble without detergents. Here we report using the QTY code to systematically replace the hydrophobic amino acids leucine, valine, isoleucine, and phenylalanine in the seven transmembrane α-helices of CCR5, CXCR4, CCR10, and CXCR7. We show that QTY code-designed chemokine receptor variants retain their thermostabilities, α-helical structures, and ligand-binding activities in buffer and 50% human serum. CCR5, CXCR4, and CXCR7 also bind to HIV coat protein gp41-120. Despite substantial transmembrane domain changes, the detergent-free QTY variants maintain stable structures and retain their ligand-binding activities. We believe the QTY code will be useful for designing water-soluble variants of membrane proteins and other water-insoluble aggregated proteins.

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The genetic code is universal, but recombinant protein expression in heterologous systems is often hampered by divergent codon usage. Here, we demonstrate that reprogramming by standardized multi-parameter gene optimization software and de novo gene synthesis is a suitable general strategy to improve heterologous protein expression. This study compares expression levels of 94 full-length human wt and sequence-optimized genes coding for pharmaceutically important proteins such as kinases and membrane proteins in E. coli. Fluorescence-based quantification revealed increased protein yields for 70% of in vivo expressed optimized genes compared to the wt DNA sequences and also resulted in increased amounts of protein that can be purified. The improvement in transgene expression correlated with higher mRNA levels in our analyzed examples. In all cases tested, expression levels using wt genes in tRNA-supplemented bacterial strains were outperformed by optimized genes expressed in non-supplemented host cells.

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