AF1501), using a 1.0 x 0.4 cm path length fluorescence cuvette at the excitation and emission wavelengths of 350 nm
and 400-550 nm, respectively.
Results and Discussion
1. Construction of L110P cDNA and induction for synthesis in
Pichia
Human L110P cDNA was successfully constructed by site-direct mutagenesis, as described in the Methods.
His
+
Mut
+
transformants were selected by growing on a minimal medium containing methanol. The
Pichia
recombinant clones were screened for the Mut
+
after they had been grown for 3 days. Twenty to fifty colonies of the
Pichia
transformants with His
+
Mut
+
phenotype were selected for the protein synthesis in small scale. By native-
PAGE, it showed that L110P was successfully synthesized and secreted out from the yeast cells into the culture
medium (Figure 1), and production of the recombinant protein reached to maximum after induction with 0.5%
methanol for 3 days. Yield of the production was ~0.5 mg/liter medium, which is less than previously reported for the
recombinant human wild type TTR (Prapunpoj
et al
., 2006).
Figure 1
Analysis of the
Pichia
transformant culture
Pichia
clone was induced to synthesize L110P with 0.5% for 7 days in 5 ml of medium, then
aliquot of the culture supernatant was analyzed by native-PAGE, followed by silver staining.
M, human plasma (2µl);1-9, individual
Pichia
transformants of L110P.
2. Purification and characterization of the recombinant L110P
Human TTR from plasma moves faster than albumin during electrophoresis at pH 8.6(Siebert and Nelson,
1942; Larsson
et al.
, 1985; Duan
et al.
, 1995; Prapunpoj
et al.
, 2000; Prapunpoj
et al.
, 2002), and it could be separated
from other proteins in yeast culture medium by preparative native-PAGE (Prapunpoj
et al
., 2006). Therefore, in this
work, the preparative native-PAGE was selected to purified the recombinant L110P from the
Pichia
culture. It showed
that TTR was effectively separated from other proteins after the culture supernatant. The eluting fractions from the
native-PAGE showed the presence of a protein band corresponding to TTR (Figure 2). In addition, it showed that the
