In Vitro Selection of Fab Fragments by mRNA Display and Gene-Linking Emulsion PCR

Figure 2

Scheme of in vitro selection of Fab fragments using mRNA display and emulsion PCR. Step 1: A randomly mutated DNA library of an H chain gene and an L chain gene is separately prepared. Each DNA library is transcribed and puromycin is ligated to the 3′ end to make an mRNA template. Step 2: The mRNA template is translated to form an mRNA-displayed molecule. Step 3: The molecules are purified and subsequently reverse is transcribed to make the mRNA portion a DNA hybrid. The H and L chain molecules are combined to form an mRNA-displayed Fab fragment. Step 4: The mRNA-displayed Fab fragments are subjected to in vitro antigen selection. Step 5: The selected mRNA-displayed Fab fragments are recovered and mixed with PCR reagents. The mixture is emulsified and the corresponding H and L chain genes are linked together by overlap extension PCR. Step 6: The linked DNA is amplified by PCR again with different primers to regenerate the H and L chain genes. Step 7: The regenerated genes are reamplified by DNA shuffling to make the templates for the next round of selection. Step 8: After a suitable number of rounds of selection, the selected DNA is cloned and sequenced to identify the selected Fab fragments.