Introduction
Amyloidosis is a group of diseases resulted from the abnormal deposition of one or several particular
aggregated proteins called “amyloids” in various tissues. Amyloids can deposit in a localized area with harmless or
only affect on a particular tissue. Up to date more than 20 amyloid precursor proteins have been identified includidng
transthyretin (TTR).
TTR is a homotetrameric protein which major functions as a thyroid hormone distributor found in human
plasma and cerebrospinal fluid (for review see Raghu
et al
., 2002). Each of TTR subunit has a molecular weight of
~15 kDa, and four of them non-covalently bind and form a hollow core containing two binding sites for L-3, 5, 3
, 5
-
tetraiodothyronine (Thyroxine; T4) and L-3,5,3
-triiodothyronine (T3). Besides, TTR also binds retinol-binding
protein (RBP), and play role in metabolism of vitamin A via its binding (for review see Lundberg
et al
., 2006). The
mutation of the TTR gene can generate an abnormal TTR monomer that easily changes in conformation and forms
amyloid fibrils naturally
in vivo
. Systemic senile amyloidosis and familial, or hereditary, amyloidoses are the two
major types of amyloidosis that are resulted from deposition of TTR amyloids. The hereditary amyloidoses that TTR
is the main cause are familial amyloidotic polyneuropathy (FAP) and familial amyloidotic cardiomyopathy (FAC).
Till now, more than 90 single point mutations have been identified in TTR gene, and approximate 70 mutations are
close related to both FAP and FAC. Amongst, TTR Val30Met (V30M) in which valine at position 30 of the TTR
polypeptide is replaced by methionine is the most frequent (Saraiva, 1984, 2001). Since the distribution and severity
of each TTR variants is depending on populations, type and characteristics of TTR variant distribute in a particular
area is essential for accomplishing the specific diagnosis and effective treatment.
Recently, a few novel single point mutations leading change in amino acid sequence of TTR monomer have
been identified. Based on the mutated position, all of them have high tendency to form amyloid, and thus, to
amyloidosis. Therefore, to accomplish for direct diagnosis and specific treatment for Thai patients, information on
type and characteristics of the TTR variant distribute in this area is essential In this research, we attempted to
synthesize TTR variant L110P by using the recombine protein expression system of
Pichia pastoris
, therefore, its
properties in particular amyloid fibril formation and association to amyloidosis can be explore.
Objective
To synthesize and characterize recombinant human L110P and to compare its fibril formation with that of
V30M
.
Methods
1. Construction of an expression vector forL110P
A site-direct mutagenesis and PCR were performed to generate and amplify the cDNA of L110P from the
cDNA of TTR wild type. The reaction involved annealing of the selection oligonucleotide, which encodes mutation
that create a resistance to the antibiotic mixture to facilitate selection of the desired mutation, and the appropriate
