Protein library display systems and methods thereof

A nucleic acid comprising, in an upstream to downstream direction:

• a first portion comprising a nucleotide sequence encoding an RNA including at least 2 boxB domains, the RNA forming hairpin structures that correspond in number to the boxB domains, wherein each hairpin structure includes a stem and a loop, and wherein each individual boxB domain is located in a separate and individual loop;
• a second portion comprising a nucleotide sequence encoding a protein including at least 2 lambda bacteriophage anti-terminator protein N domains (λ domain), wherein each individual λ domain is orientated to specifically bind to a separate and individual boxB domain;
• and
• a third portion comprising a nucleotide sequence encoding a protein of interest, wherein the third portion is operably coupled to the first portion and the second portion,
• wherein the nucleotide sequence of the second portion comprises a nucleotide sequence having at least 80% identity to SEQ ID NO:
• 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:
• 11, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:28.

A protein-RNA display construct comprising:

• a first nucleotide portion comprising an RNA including at least 2 boxB domains, the RNA forming hairpin structures that correspond in number to the boxB domains, wherein each hairpin structure includes a stem and a loop, and wherein each individual boxB domain is located in a separate and individual loop;
• a second nucleotide portion comprising an mRNA encoding a protein of interest, wherein the second nucleotide portion is coupled to the first nucleotide portion;
• a first protein portion comprising a protein including at least 2 lambda bacteriophage anti-terminator protein N domains (λ domain), wherein each individual λ domain is orientated to specifically bind to a separate and individual boxB domain;
• and
• a second protein portion comprising the protein of interest, wherein the second protein portion is coupled to the first protein portion
• wherein the RNA comprises a nucleotide sequence having at least 80% identity to SEQ ID NO:
• 14, SEQ ID NO:
• 15, SEQ ID NO:
• 16, SEQ ID NO:
• 17, SEQ ID NO:
• 18, SEQ ID NO:29, SEQ ID NO:
• 30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:
• 36, or SEQ ID NO:37.

A protein-RNA display construct comprising:

• a first nucleotide portion comprising an RNA including at least 2 boxB domains, the RNA forming hairpin structures that correspond in number to the boxB domains, wherein each hairpin structure includes a stem and a loop, and wherein each individual boxB domain is located in a separate and individual loop;
• a second nucleotide portion comprising an mRNA encoding a protein of interest, wherein the second nucleotide portion is coupled to the first nucleotide portion;
• a first protein portion comprising a protein including at least 2 lambda bacteriophage anti-terminator protein N domains (λ domain), wherein each individual λ domain is orientated to specifically bind to a separate and individual boxB domain;
• and
• a second protein portion comprising the protein of interest, wherein the second protein portion is coupled to the first protein portion
• wherein the protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO:
• 2, SEQ ID NO:4, SEQ ID NO:
• 6, SEQ ID NO:8, SEQ ID NO:
• 10, SEQ ID NO:
• 12, SEQ ID NO:
• 13, or SEQ ID NO:22.