I. iFRAG selection by bioinformatics analysis

Sequence analysis algorithms to select suitable iFRAG regions

IIa. Cloning into His-tagged bacterial expression vector

Design of PCR primers. Amplification of the fragment from a customer-provided vector and subcloning it into a selected bacterial expression vector. Screening for the positive clones. Confirmation of the integrity of the cloned DNA by sequencing with vector-specific forward and reverse primers. Preparation of the recombinant vector from 10-ml bacterial culture.

IIb. Synthesis of iFRAG coding region and cloning into His-tagged bacterial expression vector

Design of overlapping oligos covering the iFRAG coding region. Synthesis of iFRAG coding region and subcloning it into a selected bacterial expression vector. Screening for the positive clones. Confirmation of the integrity of the cloned DNA by sequencing with vector-specific forward and reverse primers. Preparation of the recombinant vector from 10-ml bacterial culture.

III. Small scale expression test

Transformation of recombinant DNA into a suitable protease-deficient E. coli strain (e.g. BL21). Small scale protein expression evaluation of 50-ml bacterial culture at two temperatures (37ºC and 16ºC). Test for the over expression of the protein of interest by SDS-PAGE, western blot with an anti-tag antibody or a specific antibody provided by the customer and/or Peptide Mass Fingerprinting by MALDI-TOF

IV. Large scale production

Growth of one liter of bacterial culture. Induction with IPTG and culture harvesting. Cell pellets will be frozen and shipped to the customer or will go to purification phase

V. Purification of iFrag in denaturing conditions

Lysis of induced cells. IMAC of cell extracts in denaturing conditions. Preparation of samples for inoculation.