HALF-LIFE EXTENSION THROUGH O-GLYCOSYLATION - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 198

pM 2 -HA, that contains the ampicillin (Amp R ) and the
neomycin (Neo R ) resistance genes and a strong promoter
of the HaMuSV virus, LTR. EPO-WT and EPO-CTP casset
genes were inserted into the cloning site of the eukaryotic
expression vector, pGT, contains the ampicillin (Amp R ),
zeocin resistance genes and a strong promoter of the
CMV virus. hGH variant genes, WT and Chimera, were
inserted into the pCI-DHFR plasmid, an eukaryotic expres-
sion vector. TSH-WT and chimeric subunits were ligated
into the expression vector pLBCMV and transfected into
COS-7 and 293 cells.
The PCR generated constructs were completely sequenced
to ensure that no errors were introduced during the PCR.

13.3.2 Expression of Chimeric Genes

Expression vectors containing wild-type or CTP-chimeric
genes were transfected into Chinese hamster ovary (CHO)
cells. Stable clones expressing hormones were selected
using antibiotics (neomycin or zeocin) and Western blot
analysis. Assembly of hFSH b -CTP with a -subunit was
examined by transfecting stable clones expressing the
a -subunit with hFSH b -CTP. Immunoprecipitation studies
prove that dimerization has occurred. It seems that a - and
b -subunits of FSH-WT migrate to the same level, meaning
that

FIGURE 13.4 Crystallographic diagram of GH and its receptor
complex. The N-terminal and C-terminal ends of GH are not
involved in the binding site of the hormone.

13.3.1 Construction of Chimeric Genes
and Expression Vectors

the molecular weight of both subunits is similar

Overlapping mutagenesis PCR technique was used to ligate
the CTP to the coding sequence of the hormone. Using this
technique, the cassette gene containing the recognition sites
of the O-linked oligosaccharides on CTP was ligated in
tandem to the coding sequence of the hormone as a single
peptide chain. It was hypothesized that hormone containing
the CTP would have a prolonged half-life and higher
bioactivity in vivo.
The FSH b wild-type (WT), FSH b -CTP, and FSH a
genes were inserted into the eukaryotic expression vector,

( 22 kDa). However, FSH-CTP migrates slower in the

gel and exhibits higher molecular weight ( 30 kDa) com-

paring to a -subunit (Figure 13.5). Similarly, The EPO-WT
migrated faster than EPO-CTP. EPO-CTP exhibited high
molecular weight

( 48 kDa) comparing to EPO-WT

( 36 kDa) (Figure 13.5). The hGH-WT migrated faster

than GH-CTPs and exhibited molecular weight of

22 kDa. GH variants contain three CTPs exhibited higher
molecular weight of

47.5 kDa. The increase of molecular
weight of the variants containing CTP is due to the addition

FIGURE 13.5 Expression of FSH, EPO, and GH chimeras from transfected CHO cells. Condition media from transfected cells were
prepared for SDS-PAGE and proteins were detected by Western blot analysis using specific antisera.