Expression and protein recovery

The context of the mRNA sequence around the AUG codon translational start site, where the ribosome initiates translation of protein synthesis, is an important consideration. An appropriate consensus ribosome-binding region for the host organism should be used and potentially inhibitory RNA secondary structure, that may affect the ability of the ribosome to access this translation start site, avoided. Various RNA structure prediction programs such as 

If a protein/peptide is secreted into the culture medium then it must be recovered from a dilute solution. This could involve precipitating the protein/peptide under nondenaturing conditions, such as using ammonium sulfate and resolubilizing in a smaller volume. Alternatively, a more convenient method is through the use of affinity materials (see below) with an appropriate matrix added to the supernatant to allow batch binding of the target protein. 

Fusion partners for recombinant protein/peptide purification  

Fusion partner Purification method Size (kDa) or aa (sequence) 

Ketosteroid isomerase (KSI) Produces insoluble fusion protein 125 aa 

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