Use of a mini-intein for the affinity purification of recombinant therapeutic proteins from Escherichia coli periplasm

Salta

Congreso; Joint LV Annual SAIB Meeting and XIV PABMB Congress.; 2019

The aim of this work was to develop a new expression and purification methodology ofrecombinant therapeutic proteins in Escherichia coli. This development combines thetargeting of the recombinant protein to the periplasmic space with the use of self-cleavable affinity tag for the subsequent purification of the recombinant protein. Theexpression of recombinant proteins directed by a periplasmic signal peptide has severaladvantages. It allows the expression of proteins with any amino acids at the N-terminal,a better folding of proteins containing disulfide bridges and facilitates the purification ofthe expressed recombinant proteins. We use human growth hormone (hGH) as modeltherapeutic protein. The therapeutic hGH need to start with a Phe residue, contain twodisulfide bridges and cannot have affinity tags. We designed an expression vector toproduce hGH N-terminal fused to the DsbA signal peptide and C-terminal fused to theSsp DnaX mini intein-Chitin Binding Domain (CBD) chimeric protein. The CBD tagallows purification of proteins by affinity chromatography while Ssp DnaX mini-inteinundergoes a self-cleavage reaction enabling the elution of rhGH without the affinity tag.The hGH coding sequences were E. coli codon optimized and synthesized with the firstnatural amino acid (Phe) right after cleavage site of signal peptide and with the lastamino acid right before cleavage site of Ssp DnaX. Optimization of the upstreamconditions allowed us the expression of six times more recombinant protein in theperiplasm than that obtained under non-optimized conditions. Then, the Ssp DnaX-CBDpolypeptide allowed the purification of the recombinant protein by affinitychromatography using a chitin column. Elution of the hGH without the affinity tag wasachieved after induction of the self-cleavage of the Ssp DnaX mini-intein by changingthe pH and temperature of the column buffer. The recombinant hGH without any tagwas efficiently recovered with high purity under these experimental conditions. Theseresults suggest that this expression system could be used for the expression andpurification of proteins or protein segments that must have their native amino acidsequence.