mobility under native condition of the recombinant L110P was similar to that of TTR in human plasma, but faster than
albumin. The analysis by SDS-PAGE of the purified recombinant TTR visualized by Coomassie blue revealed that
weight of the recombinant TTR subunit was ~16.6 kDa (Figure 3). This was slightly larger compared to human native
TTR, implying to a possible modification of the monomer.
Figure 2 Elution pattern of L110P from the preparative native-PAGE
The
Pichia
clone was induced for the synthesis of L110P for 3 days. Then, the culture supernatant
was concentrated, then loaded onto a polyacrylamide gel (12% resolving gel, 4% stacking gel) and
separated by preparative native-PAGE. The eluting fractions (2ml/fraction) were collected and 60
µl of each fraction was analyzed by native-PAGE followed by silver staining.
HP, human plasma was overloaded (2 µl) to indicate the positions of albumin and TTR in plasma;
C, concentrated culture supernatant; 1-7, individual eluting fractions.
Figure 3 Determination of subunit weight of the L110P
A plot between the relative mobility (Rf) and log of molecular weight (log MW) of protein markers
was performed and it was used in determining the subunit weight of L110P. The relative mobility
of L110P (a) was indicated.
a
