GMO and protein engineering - Enzymes in Food Technology - page 29

Forward primer based on N-terminal amino acid sequence

S. cinnamoneus
genome DNA

Reverse primer based on consensus amino acid
Sequence of bacterial SMases

PCR

Amplification and sequencing
of core region of SMase encoding gene

Amplification of 5 -flanking region

using genome DNA walking

′

Amplification of 3 ′ -flanking region

using inverse PCR

5 5

3 3

Restriction enzyme digestion

Restriction enzyme digestion

Ligation

Adapter
for genome DNA
walking

Ligation

1st PCR

Inverse PCR

Nested PCR

Sequencing of 5 ′ -flanking region Sequencing of 3 ′ -flanking region

Determination
of SMase encoding gene

Fig. 2.4 Cloning strategy of SMase encoding gene.

to improve the properties. This modification process is called 'protein engineering'. Because
of the characteristics specific to enzymes, it is not feasible to manipulate either a protein
at its conformational state or its primary structure freely without losing physical functions.
However, thanks to recombinant DNA technologies, the modification of a protein or an