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Current
Pharmaceutical Biotechnology
ISSN: 1389-2010
Current Pharmaceutical Biotechnology
Volume 11, Number 3, April 2010
Contents
Expression and Refolding Technologies for Production
of Recombinant Proteins
Guest Editor: Tsutomu Arakawa and Kouhei
Tsumoto
Editorial Pp. 231
[PMID:
20406165 PubMed - indexed for MEDLINE]
Expression
Cell Engineering and Cultivation of Chinese Hamster Ovary
(CHO) Cells Pp. 232-240
Takeshi Omasa, Masayoshi Onitsuka and Wook-Dong
Kim
[Abstract] [Purchase
Article] [PMID:
20210750 PubMed - indexed for MEDLINE]
Fusion Partners as a Tool for the Expression of Difficult
Proteins in Mammalian Cells Pp. 241-245
Jue Zhang, Jane Carter, Sophia Siu, Jason W. O’Neill,
Andrew H. Gates, John Delaney and Christopher Mehlin
[Abstract] [Purchase
Article] [PMID:
20210749 PubMed - indexed for MEDLINE]
Silkworm as a Host of Baculovirus Expression
Pp. 246-250
Akihiro Usami, Takeo Suzuki, Hidekazu Nagaya, Hiroki Kaki
and Seiji Ishiyama
[Abstract] [Purchase
Article] [PMID:
20210748 PubMed - indexed for MEDLINE]
Brevibacillus Expression System: Host-Vector
System for Efficient Production of Secretory Proteins
Pp. 251-258
Makoto Mizukami, Hiroshi Hanagata and Akira Miyauchi
[Abstract] [Purchase
Article] [PMID:
20210747 PubMed - indexed for MEDLINE]
Recombinant Expression in Moderate Halophiles Pp.
259-266
Masao Tokunaga, Tsutomu Arakawa and Hiroko Tokunaga
[Abstract] [Purchase
Article] [PMID:
20210746 PubMed - indexed for MEDLINE]
A Highly Controllable Reconstituted Cell-Free System
-a Breakthrough in Protein Synthesis Research Pp.
267-271
Hiroyuki Ohashi, Takashi Kanamori, Yoshihiro Shimizu and
Takuya Ueda
[Abstract] [Purchase
Article] [PMID:
20210745 PubMed - indexed for MEDLINE]
The Wheat-Germ Cell-Free Expression System
Pp. 272-278
Kazuyuki Takai, Tatsuya Sawasaki and Yaeta Endo
[Abstract] [Purchase
Article] [PMID:
20210744 PubMed - indexed for MEDLINE]
Development of an Insect Cell-Free System Pp. 279-284
Toru Ezure, Takashi Suzuki, Masamitsu Shikata, Masaaki
Ito, Eiji Ando, Toshihiko Utsumi, Osamu Nishimura and
Susumu Tsunasawa
[Abstract] [Purchase
Article] [PMID:
20210743 PubMed - indexed for MEDLINE]
Refolding
Step-Wise Refolding of Recombinant Proteins Pp. 285-288
Kouhei Tsumoto, Tsutomu Arakawa and Linda
Chen
[Abstract] [Purchase
Article] [PMID:
20210742 PubMed - indexed for MEDLINE]
Urea-Gradient Protein Refolding in Size Exclusion Chromatography
Pp. 289-292
Chaozhan Wang and Yan Cheng
[Abstract] [Purchase
Article] [PMID:
20210741 PubMed - indexed for MEDLINE]
The High pH and pH-Shift Refolding Technology
Pp. 293-299
Xinli Lin and Tomomi Umetsu
[Abstract] [Purchase
Article] [PMID:
20210740 PubMed - indexed for MEDLINE]
Development of an Artificial Chaperone System Based on Cyclodextrin
Pp. 300-305
Yoshihiro Sasaki and Kazunari Akiyoshi
[Abstract] [Purchase
Article] [PMID:
20210739 PubMed - indexed for MEDLINE]
Thermal-Assisted Refolding: Dilution Folding Initiated at
High Temperature Pp. 306-308
Kentaro Shiraki and Soichiro Kayano
[Abstract] [Purchase
Article] [PMID:
20210738 PubMed - indexed for MEDLINE]
Non-Denaturing Solubilization of Inclusion Bodies Pp.
309-312
Kouhei Tsumoto, Ryota Abe, Daisuke Ejima and
Tsutomu Arakawa
[Abstract] [Purchase
Article] [PMID:
20210737 PubMed - indexed for MEDLINE]
General Article
The Effects of Radix Curcumae Extract
on Expressions of VEGF, COX-2 and PCNA in Gastric Mucosa of
Rats Fed with MNNG Pp. 313-317
Bin Lu, Linfeng Yu, Lei Xu, Hanqing Chen, Lu Zhang and
Yanjun Zeng
[Abstract] [Purchase
Article] [PMID:
20210736 PubMed - indexed for MEDLINE]
Abstracts
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Article] [PMID:
20210750 PubMed - indexed for MEDLINE]
Cell Engineering and Cultivation of Chinese Hamster Ovary
(CHO) Cells
Takeshi Omasa, Masayoshi Onitsuka and Wook-Dong
Kim
Mammalian cell lines are important host cells for the industrial
production of pharmaceutical proteins owing to their capacity
for correct folding, assembly and post-translational modification.
In particular, Chinese hamster ovary (CHO) cells are the most
dependable host cells for the industrial production of therapeutic
proteins. Growing demand for therapeutic proteins promotes
the development of technologies for high quality and productivity
in CHO expression systems. The following are fundamentally
important for effective production. 1) Construction of cultivation
process. The CHO-based cultivation process is well established
and is a general platform of therapeutic antibody production.
The cost of therapeutic protein production using CHO cells
is equivalent to that using microbial culture. 2) Cell line
development. Recent developments in omics technologies have
been essential for the development of rational methods of
constructing a cell line. 3) Cell engineering for post-translational
steps. Improvement of secretion, folding and glycosylaiton
is an important key issue for mammalian cell production systems.
This review provides an overview of the industrial production
of therapeutic proteins using a CHO cell expression system.
[Back to top] [Purchase
Article] [PMID:
20210749 PubMed - indexed for MEDLINE]
Fusion Partners as a Tool for the Expression of Difficult
Proteins in Mammalian Cells
Jue Zhang, Jane Carter, Sophia Siu, Jason W. O’Neill,
Andrew H. Gates, John Delaney and Christopher Mehlin
The expression of proteins which do not express well on their
own can be enhanced by linking them to human serum albumin
(HSA) or antibody crystallizable fragment (Fc). The constructs
shown here are designed to secrete the proteins after transient
transfection of mammalian cell lines. The fusion partners
are appended to the N-terminus of the proteins and contain
a linker designed to be proteolytically cleaved. Transient
transfection and purification protocols are provided as well
as experimental results with five interleukins.
[Back to top] [Purchase
Article] [PMID:
20210748 PubMed - indexed for MEDLINE]
Silkworm as a Host of Baculovirus Expression
Akihiro Usami, Takeo Suzuki, Hidekazu Nagaya, Hiroki Kaki
and Seiji Ishiyama
While the Baculovirus Expression Vector System (BEVS) mainly
uses insect cell lines, such as Sf9 cells, the robust high
expression system using silkworm has also been developed.
We have further improved technologies for enhancement of virus
recombination, reduction of proteolytic degradation and aggregation,
and more reliable promoters. These developments made it possible
to achieve high and soluble expression of recombinant proteins.
We review here such technology developments, advantage of
using silkworm and some example applications. There are areas
where this technology can be further improved as implicated
in the end.
[Back to top] [Purchase
Article] [PMID:
20210747 PubMed - indexed for MEDLINE]
Brevibacillus Expression System: Host-Vector
System for Efficient Production of Secretory Proteins
Makoto Mizukami, Hiroshi Hanagata and Akira Miyauchi
Brevibacillus expression system is an effective bacterial
expression system for secretory proteins. The host bacterium,
Brevibacillus choshinensis, a gram-positive bacterium,
has strong capacity to secrete a large amount of proteins
(~30g/L), which mostly consist of cell wall protein. A host-vector
system that utilizes such high expression capacity has been
constructed for the production of secretory proteins and tested
for various heterologous proteins, including cytokines, enzymes,
antigens, and adjuvants.
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[Purchase
Article] [PMID:
20210746 PubMed - indexed for MEDLINE]
Recombinant Expression in Moderate Halophiles
Masao Tokunaga, Tsutomu Arakawa and Hiroko Tokunaga
A novel expression of recombinant proteins was developed using
moderate halophiles that accumulate osmolytes and hence provide
cytoplasmic environments where osmolyte-driven folding can
take place. Promoters and selection marker were developed
for high expression of foreign proteins. Examples are given
for expression of bacterial nucleoside diphosphate kinase
and human serine racemase.
[Back to top] [Purchase
Article] [PMID:
20210745 PubMed - indexed for MEDLINE]
A Highly Controllable Reconstituted Cell-Free System
-a Breakthrough in Protein Synthesis Research
Hiroyuki Ohashi, Takashi Kanamori, Yoshihiro Shimizu and
Takuya Ueda
The PURE system is a highly controllable cell-free protein
synthesis system composed of individually prepared components
that are required for protein synthesis in Escherichia
coli. The PURE system contains neither nucleases nor
proteases, both of which degrade DNA or mRNA templates and
proteins. The protein products are easily purified using affinity
chromatography to remove the tagged protein factors. The PURE
system should help to create new fields in protein research.
[Back to top] [Purchase
Article] [PMID:
20210744 PubMed - indexed for MEDLINE]
The Wheat-Germ Cell-Free Expression System
Kazuyuki Takai, Tatsuya Sawasaki and Yaeta Endo
We have made a dramatic improvement of the wheat cell-free
protein synthesis system. The first key improvement is the
method for preparation of the cell-free extract that is free
of inhibitory factors of translation reaction. Additional
improvements include a method for preparation of transcription-ready
templates by PCR, an expression vector for the cell-free system,
and the "bilayer" mode reaction method that is much
more efficient than the batch mode method and at the same
time easy to be performed by human hands and by liquid handling
machines. We review here the history of the development and
describe the protocols for the most handy "bilayer"
method and a more efficient but complicated methods. Information
on many examples and variations of the wheat cell-free protein
synthesis methods already published elsewhere is then provided
so that the readers can understand the power and potential
applications of the methods.
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[Purchase
Article] [PMID:
20210743 PubMed - indexed for MEDLINE]
Development of an Insect Cell-Free System
Toru Ezure, Takashi Suzuki, Masamitsu Shikata, Masaaki
Ito, Eiji Ando, Toshihiko Utsumi, Osamu Nishimura and
Susumu Tsunasawa
Cell-free protein synthesis systems offer production of native
proteins with high speed, even for the proteins that are toxic
to cells. Among cell-free systems, the system derived from
insect cells has the potential to carry out post-translational
modifications that are specific to eukaryotic organisms, as
occurs in the rabbit reticulocyte system. In this review,
we describe development of this insect cell-free system and
its applications.
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[Purchase
Article] [PMID:
20210742 PubMed - indexed for MEDLINE]
Step-Wise Refolding of Recombinant Proteins
Kouhei Tsumoto, Tsutomu Arakawa and Linda
Chen
Protein refolding is still on trial-and-error basis. Here
we describe step-wise dialysis refolding, in which denaturant
concentration is altered in step-wise fashion. This technology
controls the folding pathway by adjusting the concentrations
of the denaturant and other solvent additives to induce sequential
folding or disulfide formation.
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[Purchase
Article] [PMID:
20210741 PubMed - indexed for MEDLINE]
Urea-Gradient Protein Refolding in Size Exclusion Chromatography
Chaozhan Wang and Yan Cheng
Protein refolding using urea gradient size exclusion
chromatography (UGSEC) is a new version of conventional SEC
refolding, which incorporates urea gradient into the SEC.
Operating factors, mainly the urea gradient length and the
final urea concentration in the gradient, are discussed in
detail. Recent applications of UGSEC, including refolding
of recombinant human granulocyte colony stimulating factor
by UGSEC, is shown as an example.
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[Purchase
Article] [PMID:
20210740 PubMed - indexed for MEDLINE]
The High pH and pH-Shift Refolding Technology
Xinli Lin and Tomomi Umetsu
We have developed a high-pH, pH-shift refolding “Ph-Fold
technology”, both for academic research and industrial
protein drug development applications. Using this technology,
we were able to refold some “difficult-to-refold”
proteins, some of which are proven important drug targets
and protein drug candidates. The technology is composed of
the initial E. coli production of inclusion bodies,
the high pH solubilization/pH shift refolding screening technology,
which includes automated pH shift refolding screening, and
the methods of testing protein refolding without functional
assay. This technology, especially the automated refolding
system based on the Ph-Fold technology, may help both academic
and industrial research in broadening structural genomic research,
functional proteomics, and genomic scale drug development
efforts.
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[Purchase
Article] [PMID:
20210739 PubMed - indexed for MEDLINE]
Development of an Artificial Chaperone System Based on Cyclodextrin
Yoshihiro Sasaki and Kazunari Akiyoshi
Molecular chaperones in living systems inspired us to explore
new concepts for assisting protein refolding. The chaperone
selectively interacts with a non-native protein by hydrophobic
interaction to prevent irreversible aggregation and releases
the protein in its refolded form with the aid of ATP and another
co-chaperone. Cyclodextrins have been used to simulate the
function of the chaperones by controlling the hydrophobic
interaction with proteins. In this chapter, we review the
cyclodextrin (CD)-related protein refolding systems.
[Back to top]
[Purchase
Article] [PMID:
20210738 PubMed - indexed for MEDLINE]
Thermal-Assisted Refolding: Dilution Folding Initiated at
High Temperature
Kentaro Shiraki and Soichiro Kayano
Protein refolding from unfolded state is usually carried out
at low temperature to reduce protein aggregation and proteolytic
degradation. This review briefly introduces a unique method
for the protein refolding via high temperature, typically
at above melting temperature of the protein.
[Back to top]
[Purchase
Article] [PMID:
20210737 PubMed - indexed for MEDLINE]
Non-Denaturing Solubilization of Inclusion Bodies
Kouhei Tsumoto, Ryota Abe, Daisuke Ejima and
Tsutomu Arakawa
It has been a conventional notion that cytoplasmic recombinant
expression leads to either soluble protein or inclusion bodies.
In the latter case, it was always assumed that proteins in
inclusion bodies (IBs) are more or less unfolded and hence
require complete denaturing condition for solubilization,
which uses strong detergents, urea or guanidine hydrochloride.
However, we often observe distribution of expressed proteins
in both soluble and insoluble fractions. In such expression,
IBs are often loose and of flocculate morphology. We believe
that such distribution is due to association of near native
structures of the expressed proteins, which cause either aggregation
into insoluble fractions or unstable soluble proteins. In
our experience, although not reported by others, interleukin-1α,
interferon-γ,
tumor necrosis factors, fibroblast growth factors, His-tagged
fyn kinase and many other proteins showed such behavior. If
this occurs, we have experienced problems of instability,
low yield and insolubility whether purification is done from
the soluble fraction or by refolding of IBs. Arginine has
shown great promise in non-denaturaing solubilization of some
of these proteins we have tested.
[Back to top]
[Purchase
Article] [PMID:
20210736 PubMed - indexed for MEDLINE]
The Effects of Radix Curcumae Extract
on Expressions of VEGF, COX-2 and PCNA in Gastric Mucosa of
Rats Fed with MNNG
Bin Lu, Linfeng Yu, Lei Xu, Hanqing Chen, Lu Zhang and
Yanjun Zeng
Aim: To study the effects of an extract solution
from radix curcumae on the expressions of VEGF, COX-2 and
PCNA in gastric mucosa of rats during carcinogenesis induced
MNNG.
Methods: Eighty male Wistar rats were randomly
divided into 5 groups: group A, water and normal saline; group
B, MNNG and normal saline; group C, MNNG and celecoxib; group
D, MNNG and low-dose (1g/ml) radix curcumae steam distillation
extract solution; group E, MNNG and high-dose (2g/ml) radix
curcumae wet distillation extract solution. In the end of
the 40th week, all rats alive were sacrificed and the expressions
of VEGF, COX-2 and PCNA in gastric mucosa were determined
by immunohistochemistry.
Results: The expression levels of VEGF and
COX-2 and the optical density levels of PCNA in group C, D
and E were remarkably lower than that of group B (P<0.05),
while the optical density levels of PCNA in group E were higher
than that of group C and D (P<0.05).
Conclusions: The distilled extract of curcumae
can down-regulate the expressions of VEGF, COX-2 and PCNA
in the gastric mucosa of rats during carcinogenesis induced
MNNG and can reduce the incidence of gastric caner, suggesting
it maybe as a potential chemopreventive agent for gastric
cancer.
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