Expression of synthetic genes coding for completely new, nutritionally rich, artificial proteins

Synthetic genes (A, AB and AHB) constructed and cloned into pKK233-2 vector were recloned from the parent plasmid into the new procaryotic expression vectors pGFY221N and pBI052. Gene AF-B (coding for all amino acids besides phenylalanine) was obtained by 'cassette mutagenesis' from gene AB. The plasmid pGFY221N was constructed from pGFY218L by replacing the PstI by an NcoI site; plasmid pBI052 was derived from pGFY221N through replacing the 221-bp EcoRI/NcoI fragment with a synthetic DNA segment of 52 bp representing the Escherichia coli atpE gene translational initiation region. The genes A, AB, AHB and AF-B in the vector pGFY221N were expressed with a six-amino-acid-long leader sequence; in pBI052 the genes were expressed directly. In vitro expression experiments were successfully with all the genes except with the AHB gene integrated into pGFY221N. In the E. coli minicell system expression was demonstrated with the A gene in pGFY221N and the AF-B and AHB genes in pBI052. Complete translation of the expressed genes AB, AF-B and AHB in either the in vitro or in vivo systems could be shown by using 35S-labelled N-terminal methionine and C-terminal cysteine. Both amino acids occur only once in the peptide sequences.