Abstract: The present invention relates to DNA construct suitable for expressing a recombinant protein in a bacterial host cell wherein said DNA construct comprises nucleotide sequences encoding: a rhaBAD promoter, a RhaR transcription activator, a RhaS transcription activator, an antibiotic resistance marker, a promoter operably linked to the nucleic acid encoding for the antibiotic resistance marker, an rmB T1 terminator, an rmB T2 terminator and a pMB1 origin of replication. The DNA construct may also comprise a nucleotide sequence encoding said recombinant protein operably linked to the rhaBAD promoter. The present invention further relates to a vector and bacterial host cell comprising said DNA construct as well as a method of producing said recombinant protein by exposing said bacterial host cell to rhamnose and thereby inducing expression of said recombinant protein.

Abstract: The present invention relates to DNA constructs comprising a novel TIS sequence. The TIS sequence transcribes into an RNA motif that functions as the protein translation initiation site in an mRNA transcript. The DNA construct may further comprise a nucleic acid sequence encoding signal peptide. Additionally, the DNA constructs may also comprise a nucleic acid sequence encoding a recombinant protein or one or more polypeptide chains thereof. The invention further relates to an expression vector, expression cassette and host cell which comprise said DNA constructs. Furthermore, the present invention relates to a recombinant protein expressed by the host cell as well as a method for expressing the recombinant protein.

Abstract: A method for optimising a nucleotide sequence for protein expression in a host cell, comprising the steps of: (a) constructing an expression library comprising a large number of variants of the sequence to be expressed operatively cloned in an expression vector, wherein (i) the sequence of the 6 nucleotides immediately upstream (5?-direction) of the first codon of the coding sequence to be expressed is completely or partially randomized; (ii) the sequence of the second and third codons of the coding sequence to be expressed is randomized, wherein the randomization of the second and third codons is limited to changes not altering the amino-acids encoded by said codons; (b) screening the library with regard to efficiency of protein expression in the host cell; and (c) selecting a sequence resulting in a desired level of efficiency of protein expression.

Abstract: A method for optimising a nucleotide sequence for protein expression in a host cell, comprising the steps of: (a) constructing an expression library comprising a large number of variants of the sequence to be expressed operatively cloned in an expression vector, wherein (i) the sequence of the 6 nucleotides immediately upstream (5?-direction) of the first codon of the coding sequence to be expressed is completely or partially randomized; (ii) the sequence of the second and third codons of the coding sequence to be expressed is randomized, wherein the randomization of the second and third codons is limited to changes not altering the amino-acids encoded by said codons; (b) screening the library with regard to efficiency of protein expression in the host cell; and (c) selecting a sequence resulting in a desired level of efficiency of protein expression.