Journal of Cell and Molecular Biology - ResearchGate

38 Hassan M. KHACHFE and David ATKINSON
from the particle by trypsin digestion (Yang et al.,
1989).
Because of the size and insolubility of apo B,
determination of its structural motifs responsible
for the lipid association has been so difficult that
only indirect probing has been done on this
nonexchangeable protein. Biochemical and
biophysical techniques, as well as computer
algorithms have been used to study the domain
structure and rearrangements of apo B. These
studies have deepened our understanding of the
domain arrangement of this huge protein.
Therefore, it is necessary to study the structure
of the protein in pieces, perhaps corresponding to
structural or functional domains. For this reason,
genetically engineered truncated forms have been
obtained to study the domain organization in the
protein. The N-terminal portion of the protein
posed as an interesting candidate for structural
studies for several reasons:
1) The striking homology it shows with other lipid
transporting proteins, e.g., lipovitellin, whose
structure was solved and studied vis-à-vis its
function, and therefore, opened the door for
computer modeling of the structure of apo B (Al-
Ali and Khachfe, 2007).
2) This portion of the protein shows an optimal
interaction with the microsomal triglyceride
transfer protein (MTP). The presence of MTP
complexed to the protein disulfide isomerase (PDI)
found in the endoplasmic reticulum is an absolute
requirement for the assembly of neutral lipids and
phospholipids into chylomicrons and VLDL
particles (Hussain et al., 1997).
3) Although truncated, this part of the protein is
readily associated with a variety of phospholipids
to from large discoidal particles (Herscovitz et al.,
2001), and has interesting metabolic behaviors
based on its glycosylation state, such that the
Q158N mutation of the single glycosylation site in
this domain decreases the secretion of the protein,
but has little effect on its synthesis or its
intracellular distribution (Vukmirica et al., 2002).
4) As mentioned above, the fact that seven out of
the eight disulfide bonds found in apo B100 are
located in the N-terminal domain suggest that this
portion is compact, highly organized, and most
likely globular (Prassl and Laggner, 2009).
Hence, the 17% N-terminal domain of apo B100 was
expressed with an aim to later characterize its
structure and eventually relate to its function in the
full-length protein. One of the restriction enzymes
used to cut the apo B100 gene yielded a portion that
corresponds to the 17% N-terminal part of the
protein (Herscovitz et al., 1991). Following the
same nomenclature process that described the
different truncated forms of apo B100, this portion of
the protein that corresponded to the N-terminal
17% of the full-length protein was then called apo
B17, or simply B17.
Materials and methods
Materials
Murine mammary carcinoma cells (C127)
overexpressing B17 were obtained from Dr. V.
Zannis (BUMC, Medicine) (Cladaras et al., 1987).
Sf9 cells were from Life Technologies
(Gaithersburg, MD). Baculovirus particles cloned
with the B17 gene were a kind gift from Dr. G.
Carraway. Dulbecco’s modified Eagle’s medium
(DMEM), Sf-900 II serum-free medium (SFM),
bovine fetal serum (BFS), penicillin / streptomycin
(PS), and trypsin-EDTA were from Life
Technologies (Gaithersburg, MD). N-Acetyl-Lleucinyl-L-leucinyl-L-norleucine
(ALLN),
aprotinin, leupeptin, phenyl-methyl-sulfonylfluoride
(PMSF), sodium azide, and ethylenediamine
tetraacetate (EDTA) were from Sigma (St.
Louis, MO). Broad range protein marker (6,500 –
200,000 MW) was from BioRad (Hercules, CA).
Gelatin Sepharose and Protein-G Sepharose 4
Fast Flow were from Pharmacia Amersham
(Piscataway, NJ). Polyclonal goat anti-human apo
B IgG, alkaline phosphatase-conjugated rabbit antigoat
IgG, and horse-radish peroxidase (HRP)conjugated
rabbit anti-goat IgG were from
BioDesign (Saco, ME). Polyclonal sheep antihuman
apo B IgG was from Roche Molecular
Biochemicals (Indianapolis, IN).
C-127 cells were permanently transfected with
the gene coding for B17 as previously described
(Claderas et al., 1987; Herscovitz et al., 1991).
Mass expression of the protein was achieved using
roller bottles (Claderas et al., 1987) or a Verax
System-1 Bioreactor (Verax, Lebanon, NH) that
was modified – in-house – and coupled with a
ceramic core reactor, which eventually increased
the number of cells to near tissue density while
automating the media feed and harvest processes.
The harvested or stored media were then
vacuum-filtered through 0.45 µm pore size filter
paper, and then concentrated 25-fold using an
Amicon stirred-cell with a 30,000 MWCO
membrane. The concentrate was processed for
protein purification.
Sf9 – Spodoptera frugiperda – insect cells
adapted to serum-free suspension culture in Sf-900