117
Recombinant protein expression for therapeutic applications
Dana C Andersen* and Lynne Krummen†
In recent years, the number of recombinant proteins used for
therapeutic applications has increased dramatically. Many of
these applications involve complex glycoproteins and
antibodies with relatively high production needs. These
demands have driven the development of a variety of
improvements in protein expression technology, particularly
involving mammalian and microbial culture systems.
Addresses
Cell Culture & Fermentation Research & Development, Genentech, Inc.,
1 DNA Way, South San Francisco, CA 94080, USA
*e-mail: andersen@gene.com
† e-mail: krummen.lynne@gene.com
Current Opinion in Biotechnology 2002, 13 :117–123
0958-1669/02/$ —see front matter
© 2002 Elsevier Science Ltd. All rights reserved.
Abbreviations
CHO
Chinese hamster ovary
BHK
baby hamster kidney
Introduction
As the biotechnology industry has rapidly expanded in
recent years, the expression of a spectrum of recombinant
proteins in different systems for a wide variety of purposes
has been a major feature and challenge. In some applications, a large array of proteins are needed in relatively
small quantities for screening applications, whereas in
other cases, quantities approaching the metric ton scale are
needed for specific therapeutic applications. The majority
of therapeutic proteins have been produced in either
mammalian cell-culture systems, with Chinese hamster
ovary (CHO) cells representing the most common system,
or in Escherichia coli [1,2•]. A variety of alternative expression systems are also being developed and evaluated. It is
not at all clear which of these systems will ultimately be
the most useful for the variety of niches and applications of
therapeutic protein production.
Several excellent reviews have provided comprehensive
coverage of various aspects of therapeutic protein production, including specific issues related to large-scale cell
culture production [1] and considerations for the production of specific classes of molecules such as recombinant
antibody-related products [3]. This review will focus on
selected results across a range of expression systems
(although focusing primarily on mammalian and E. coli
cell-culture systems) that illustrate important ways in
which expression technology is evolving to meet the
spectrum of research, development and commercial needs.
Rather than provide a comprehensive review of the
subject, this review will highlight several specific, recent
results across the field.
Mammalian expression systems
Over the past seven to eight years there has been considerable progress in fine-tuning mammalian expression
systems for high-level recombinant gene expression. CHO
and NS0 murine myeloma cell expression systems have
now established themselves as the predominant systems of
choice for mammalian expression. Refinements of vector
construction, choice of selectable markers and advances in
gene-targeting and high-throughput screening strategies
have made the establishment of recombinant cell lines
with high specific productivities (20–60 pg/cell/day for
antibody cell lines) relatively common and have reduced
the time required for cell line development. Recent
advancements in expression technologies using traditional
viral-promoter-based expression vectors include the development and refinement of dicistronic expression strategies
using either internal ribosome entry site (IRES) sequences
[4–7] or alternative splicing [8,9]. These strategies are
designed to link expression of the gene of interest to
the selectable marker. In addition, early reports on the
development of host cell lines that express trans-activating
factors or include cis-acting elements on expression
constructs [10••] promise further advances in the augmentation of promoter activity in the near future.
A variety of novel promoter systems are also being developed and evaluated both for recombinant drug product
expression and for directed metabolic engineering
approaches (see [11]). For example, both tetracycline- and
streptogramin-based gene regulation systems have recently
been demonstrated to be highly useful for regulatable
expression in CHO and other mammalian cell cultures
[12••,13]. These systems promise to be useful for multicomponent control strategies and for the expression of
products which themselves promote cytostasis or cytotoxicity.
For the alternative application of expressing high numbers of
proteins for screening applications, an alphavirus-based system has been reported [14]. Transient systems, in which the
isolation of stable transfectants is bypassed so that protein
expression is obtained rapidly but only for a limited period of
time, have also been shown capable of producing reasonable
protein levels (10–20 mg/L) in reduced times [15]. These
systems are providing an increasing array of tools to enable
the manipulation of large panels of genes efficiently.
One of the most notable advances in recent years has been
the application of genetic engineering approaches to rationally modify specific features of mammalian host cells to
improve their utility in recombinant protein expression
applications. One such example is in the area of glycosylation control. Work over the past decade has demonstrated
how various reactions in the glycosylation pathway can be
influenced by cell-culture factors, host-cell selection and
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Biochemical engineering
protein-specific features (reviewed in [16,17,18•]), leading
to the production of molecules with variable or suboptimal
clearance or bioactivity properties. Recent studies have
shown that specific manipulation of oligosaccharide
structures on a recombinant protein by overexpression of
appropriate glycosyltransferases can enhance glycan
quality. Improvements are made either by increasing the
homogeneity of native structures or by introducing nonhost-cell residues to specialize glycan quality and function.
For example, the overexpression of a galactosyltransferase
and a sialyltransferase in CHO cells led to corresponding
increases in the galactose and sialic acid content of
expressed recombinant therapeutic proteins [19••]. Other
groups have successfully overexpressed N-acetylglucosaminyltransferase III to increase the fraction of bisecting
N-acetylglucosamine residues on antibodies produced in
CHO cells [20,21]. Sialic acid has also been introduced in
an α2,6-linkage to glycoproteins synthesized by CHO and
baby hamster kidney (BHK) cells that lack the specific
sialyltransferase responsible for this transfer [18•,22,23].
Another focus of genetic manipulation has been to improve
the efficiency of central carbon metabolism and to reduce
lactate accumulation. Earlier work showed that overexpression of a pyruvate carboxylase gene in BHK cells reduced
lactate accumulation and improved cell yields on glucose,
although it is not clear if this approach would be helpful in
CHO cultures [24]. In an alternative approach, partial
disruption of the gene encoding lactate dehydrogenase A
(LDH-A) led to reduced lactate and improved cell-culture
performance in hybridoma cultures [25].
Perhaps the most interesting of the targets for metabolic
control have been highlighted by recent studies to control
cell growth and to limit apoptotic cell death. Several
previous studies have shown that recombinant protein
production may be linked to the growth state of the
culture. These studies have shown both growth-associated
and inverse growth-associated production in different
systems. Recent attempts to exploit this phenomenon
have used inducible expression of the product gene in
concert with a regulatory protein (such as p27) that induces
cell-cycle arrest at the G1/S phase border. These studies
support the hypothesis that growth arrest, at least in this
case, is associated with as much as a 15-fold increase in
specific productivity [26,27••]. Increased productivity has
also been observed in response to temperature shift and in
BHK cultures in which cell proliferation was controlled by
expression of the tumor suppressor IRF-1 (interferon
regulatory factor 1) [27••,28,29]. In both instances, however,
direct effects of temperature and IRF-1 on transcriptional
and/or translational control may play a role. Whether or not
growth control can be balanced with enhancements in
cell-specific productivity in such a way as to significantly
enhance volumetric productivity remains to be seen.
Although the potential benefits of these approaches are
encouraging, questions remain regarding the general application of the specific promoter systems, cell-line-specific
effects and the method of growth control in controlling
growth-related productivity effects (see [30–32]).
Similarly, work demonstrating the ability of chemical
inhibitors of apoptosis to extend culture viability has laid
the foundation for a growing area of work directed towards
genetic engineering of cell death pathways. Overexpression
of Bcl-2 and BclxL has been shown to inhibit apoptotic
death in several different mammalian culture systems (see
[33,34]). A recent report also showed increased volumetric
antibody expression in butyrate-treated CHO cultures
overexpressing Bcl-2 [35]. Although promising, the effects
observed in these studies have been relatively modest and
variable depending on the cell line and the apoptotic stimuli.
This is perhaps not surprising given the complexity and
redundancy of the regulatory pathways involved in apoptosis, and multifaceted approaches may be required to
achieve significant benefits. Researchers are now expanding their approaches to include inhibitors of caspase
activation and mitochondrial cytochrome c release. A recent
genetic engineering study using overexpression of Bcl-2
deletion mutants in CHO and BHK cells also suggested
that the inherent susceptibility of wild-type Bcl-2 to intracellular degradation may explain some of the limited effects
on productivity that have been reported for Bcl-2-based
strategies [34,36••].
In addition to genetic approaches, several recent studies
show that environmental control strategies continue to be
useful for manipulating glycosylation as well as carbon
metabolism, cell growth and cell death. For instance,
medium supplementation with the nucleotide sugar precursors glucosamine and N-acetylmannosamine enabled
some level of glycosylation control of a recombinant glycoprotein produced in NS0 and CHO cultures [37,38].
Additionally, advancements in fed-batch and batch media
compositions have dramatically improved viable cell density and the duration of serum-free cell culture through a
general retardation of onset of apoptosis (e.g. [39]).
Likewise, reduced temperatures have been shown to prolong cell viability and, in some cases, enhance cell-specific
productivity either through growth arrest phenomenon or
through the modulation of cold-sensitive gene expression
[27••]. Other studies have identified media additives
directed specifically towards the inhibition of apoptosis
[40,41]. In a related study, rapomycin was fed to hybridoma
cultures in an effort to reduce cell death via cell-cycle
modulation, yet was found to have a broadly beneficial
effect on growth and productivity in this system [42].
Microbial expression systems
The primary microbial host for producing recombinant
therapeutic proteins has been E. coli, and recent reviews
have provided excellent summaries of key elements of this
topic [2,43].
In many cases, the production of soluble, active protein is
desired (as opposed to inclusion-body formation), and a
Recombinant protein expression for therapeutic applications Andersen and Krummen
variety of cases where proteins with chaperone-like activity
have enhanced folding have been recently reported. DegP
represents one interesting case as it has been demonstrated
to have chaperone-like activity in at least two cases at
reduced temperature, along with its known protease activity
at higher temperatures [44,45]. Recent work has shown
that mutants with modulated activities of the disulfide
oxidoreductases DsbA and DsbC can be used to enhance
disulfide isomerization and protein folding in the
periplasm [46]. These results complement earlier work
showing the dramatic improvements in yields that can be
achieved in some cases by overexpressing these proteins
[47]. Overexpression of the periplasmic peptidylprolyl
isomerase FkpA has been shown to increase yields of a
single-chain Fv fragment (scFv), although the effect is
apparently independent of the peptidylprolyl isomerase
activity [48]. In an alternative approach, Fab antibody
fragments were expressed in the cytoplasm of trxB mutants
in which a variety of chaperones were co-expressed and
seen to have effects on folding [49••]. ClpG and HtbG
have also been shown to facilitate protein folding in E. coli
[50], a result that highlights the beneficial role that stress
proteins can have in protein folding. Collectively, these
results demonstrate the utility of co-expressing specific
factors to modulate the folding environment.
Although the various promoter systems commonly used for
E. coli have been well described [43], several recent reports
have demonstrated new options. In one particularly notable
result, secondary structure was engineered into mRNA transcripts to manipulate message stability and, correspondingly,
expression level [51••]. This provides another technique,
along with manipulation of translation initiation strength via
the ribosome-binding site [52], for controlling the relative
strength of expression of sets of genes independently. Other
recent reports have developed and demonstrated useful
protein expression systems for different applications via the
araE, rhaBAD and nar promoters, which use arabinose,
rhamnose and microaerobic conditions, respectively, to
induce recombinant protein expression [53–55].
As in the case of mammalian systems, much recent work
has focused on metabolic factors that might more broadly
improve the effectiveness of recombinant E. coli for
protein production in high cell density fermentors.
Historically, acetate accumulation has provided perhaps
the best example of such an issue for E. coli, and a variety
of potential solutions have been reported (see [2•]). Recent
strategies to address this issue include the development of
more sophisticated glucose feeding strategies to minimize
acetate accumulation and the use of a pyruvate kinase
mutant [56,57]. The use of E. coli expressing Vitreoscilla
hemoglobin to improve growth (and product yield) in
microaerobic fermentor environments has also been further
developed. Mutagenesis techniques have been used to
generate improved hemoglobins for this task, and other
bacteria have been screened for alternative hemoglobinlike proteins [58,59]. Other studies have used techniques
119
such as nuclear magnetic resonance (NMR)-based flux
analysis to address questions regarding the mechanism of
the hemoglobin benefit [60,61].
Recombinant protein expression has also been shown to
elicit a variety of stress responses in E. coli, some of which
were recently evaluated using DNA microarrays [62]. These
stress responses, in turn, may be highly beneficial for recombinant protein expression; for example, the presence of
stress-induced proteins was recently shown to increase the
folding and formation of soluble human fibroblast growth
factor 2 [63]. A strategy of inducing various stress responses
before recombinant protein expression has been recently
evaluated and revealed one case where the addition of
dithiothreitol 20 min before induction of a green fluorescent
protein–chloramphenicol acetyltransferase fusion protein
led to increased activity of the model protein following
induction [64]. Another report investigated the role that FIS
(a DNA-binding protein implicated in stimulating ribosome
synthesis) overexpression might have in increasing heterologous protein expression [65]. Finally, the possibility of a
role for quorum-sensing mechanisms and the characterization of complex metabolic oscillations in recombinant E. coli
fermentations highlight the complexity and the continuing
potential for surprises [66,67]. In a different study, overexpression of the rspAB operon (involved in repression of
σs-regulated proteins) led to improved recombinant protein
production, presumably via a homoserine lactone-dependent mechanism; however, the possibility of cell-to-cell
signaling effects was also raised in this study [68].
With the increasing volume of results from E. coli systems, particularly as a result of the application of genomic and proteomic
technologies, the utility of mathematical frameworks to integrate and analyze this data has become more apparent. Recent
models addressing this issue have been described both at the
whole-cell level [69,70] and for specific elements such as
protein folding and aggregation [71] and stress responses [72].
Other expression systems
Both transgenic plants and animals, as well as plant tissue
cultures, have been used to produce a variety of different
recombinant proteins (reviewed in [73–75]). Whereas
limits in glycosylation have been cited as one challenge for
recombinant protein production in plants, the problem of
proteolysis was also evaluated in a recent report on the
potentially important application of antibody production in
plant culture [76••].
Insect cells have been used in a variety of protein expression
applications, particularly related to high-throughput expression of sequences for functional screening. One recent major
focus has been on attempts to generate proteins with fully
sialylated, complex oligosaccharides — a topic of some historical controversy (see [77]). Overexpression of appropriate
galactosyltransferases and sialyltransferases has led to some
success in generating sialylated oligosaccharides on insectderived proteins. It appears, however, that other limitations
120
Biochemical engineering
might also be important for the consistent production of
highly sialylated proteins [78,79,80••,81,82]. These include
the deficiency of the N-acetylglucosaminyltransferase II
enzyme responsible for initiation of the second complextype antennae of the oligosaccharide structure, as well as the
production of the cytidine monophosphate-NeuAc precursor
and host-specific effects.
Yeast systems, of which the two major systems are
Saccharomyces cerevisiae and Pichia pastoris, are also being
pursued. The use of P. pastoris for the production of recombinant human chitinase for clinical studies was recently
described in a perfusion system, yielding in excess of
300 mg/L/day [83]. P. pastoris has also been utilized in a
fed-batch system to produce an insulin precursor at yields
of 1.5 g/L [84]. Other reports have investigated more
complex feeding strategies for these systems and include a
case for the production of a recombinant scFv antibody
fragment [85,86]. Recent studies with S. cerevisiae have
explored both the strong cell-cycle dependence and metabolic burden of heterologous protein secretion in these
cultures. This work provides both interesting points for
comparison with analyses in microbial and mammalian systems, as well as an improved fundamental understanding of
the use of yeast for recombinant protein production [87,88].
The efficiency of cell-free expression systems also continues
to improve, both for more traditional systems using cell
extracts and for systems reconstituted from purified
components [89,90••] (reviewed in [2•]). Although currently
useful for high-throughput screening applications, it will
be interesting to see in coming years to what extent these
systems could be useful for larger scale production needs.
Linking expression and purification
Increasing attention has been paid to optimizing expression
systems in the context of the production process to aid in
protein recovery and to improve overall yields and efficiencies. For example, the complementary expression of
holin and lysozyme enzymes in E. coli to facilitate the
release of products has been demonstrated for two different cases [91–93] The co-expression of an endonuclease
was also used to improve the properties of the lyzed
fermentation broth for subsequent processing [91]. A very
thorough demonstration of the integration of fermentation
and recovery was reported for human chymotrypsin B production in P. pastoris [94•,95]. In this analysis, high-density
fed-batch fermentations and a lower density continuous
process were evaluated with alternative recovery procedures, including two-phase extraction and expanded-bed
chromatography, to develop an integrated process solution.
In another report, a new affinity tag to provide additional
means for efficient purification of the protein of interest
was demonstrated [96].
Conclusions
In recent years, a variety of different platforms have been
investigated for the production of recombinant therapeutic
proteins. CHO and NS0 murine myeloma cells are the
predominant mammalian cell lines used, and recent progress
has been made in promoter systems and in the manipulation
of these cell lines to improve glycoprotein production in
fermentors. Rational engineering of E. coli has also continued, with notable advancements in improving the cellular
environment for protein folding. Progress has been made in
addressing the potential limitations of a variety of other
systems including yeast, insect and plant culture systems.
One general trend across these platforms has been an expansion in the focus of study. Whereas in the past work has
focused specifically on heterologous product protein expression, it has now expanded to include efforts to improve
global cellular properties to enable more efficient and effective production in industrial environments. Another recent
trend has been an increased focus on integrated bioprocessing
approaches, such as the development of features in expression systems specifically targeted towards improving
recovery and purification of the protein of interest.
Acknowledgements
The authors would like to thank John Joly, Brad Snedecor and Gian Polastri
for discussions and critical review of the manuscript.
References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
• of special interest
•• of outstanding interest
1.
Chu L, Robinson DK: Industrial choices for protein production by
large-scale cell culture. Curr Opin Biotechnol 2001, 12 :180-187.
2. Swartz JR: Advances in Escherichia coli production of therapeutic
•
proteins. Curr Opin Biotechnol 2001, 12 :195-201.
This review provides a more comprehensive analysis of recent developments
in recombinant protein expression, involving E. coli in particular.
3.
Chadd HE, Chamow SM: Therapeutic antibody expression
technology. Curr Opin Biotechnol 2001, 12 :188-194.
4.
Gertu V, Yan G, Zhang G: IRES bicistronic expression vectors for
efficient creation of stable mammalian cell lines. Biochem Biophys
Res Commun 1996, 229 :295-298.
5.
Pu H, Cashion LM, Kretschemer PJ, Liu Z: Rapid establishment of
high-producing cell lines using dicistronic vectors with glutamine
synthase as the selection marker. Mol Biotechnol 1998, 10 :17-25.
6.
Mielke C, Tummler M, Schubeler D, von Hoegen I, Hauser H:
Stabilized, long-term expression of heterodimeric proteins from
tricistronic mRNA. Gene 2000, 254 :1-8.
7.
Hennecke M, Kwissa M, Metzger K, Oumard A, Kroger A,
Schirmbeck R, Reimann J, Hauser H : Composition and arrangement
of genes define the strength of IRES-driven translation in
bicistronic mRNAs. Nucleic Acids Res 2001, 29 :3327-3334.
8.
Lucas BK, Giere LM, Demarco RA, Shen A, Chisholm V, Crowley CW :
High level production of recombinant proteins in CHO cells using
a dicistronic DHFR intron expression vector. Nucleic Acids Res
1996, 24 :1774-1779.
9.
Werner RG, Noe W, Kopp K, Schulter M: Appropriate mammalian
expression systems for biopharmaceuticals. Drug Res 1998,
48 :870-880.
10. Zahn-Zabal M, Kobr M, Girod P-A, Imhof M, Chatellard P, de Jesus M,
•• Wurm F, Mermod N: Development of stable cell lines for
production or regulated expression using matrix attachment
regions. J Biotechnol 2001, 87 :29-42.
This report compares the ability of several chromatin elements associated
with defining transcriptional control regions to provide enhancement of
stable or regulated gene expression in CHO cells when included on the
expression plasmid or as a co-transfected element. The authors identify the
Recombinant protein expression for therapeutic applications Andersen and Krummen
chicken lysozyme matrix attachment region as superior in enhancing expression
of a reporter gene or model antibody.
11. Fussenegger M: The impact of mammalian gene regulation
concepts on functional genomics research, metabolic
engineering, and advanced gene therapies. Biotechnol Prog 2001,
17 :1-51.
12. Fussenegger M, Morris RP, Fux C, Rimann M, von Stockar B,
•• Thompson CJ, Bailey JE: Streptogramin-based gene regulation
systems for mammalian cells. Nat Biotechnol 2000, 18 :1203-1208.
This report describes a novel regulated gene expression control system
based on the concepts previously used by the same group to develop the
tetracycline-repressed gene expression system, Tet-off. This system enables
dual control of critical regulatory genes (see also [13]).
13. Fux C, Moser S, Schlatter S, Rimann M, Bailey JE, Fussenegger M:
Streptogramin- and tetracycline-responsive dual regulated
expression of p27(Kip1) sense and antisense enables positive
and negative growth control of Chinese hamster ovary cells.
Nucleic Acids Res 2001, 29 :E19.
14. Koller D, Ruedl C, Loetscher M, Vlach J, Oehen S, Oertle K,
Schirinzi M, Deneuve E, Moser R et al.: A high-throughput
alphavirus-based expression cloning system for mammalian cells.
Nat Biotechnol 2001, 19 :851-855.
15. Meissner P, Pick H, Kulangara A, Chatellard P, Friedrich K, Wurm FW :
Transient gene expression: recombinant protein production with
suspension-adapted HEK293-EBNA cells. Biotechnol Bioeng
2001, 75 :197-203.
16. Goochee CF, Gramer MJ, Andersen DC, Bahr JB, Rasmussen JR:
The oligosaccharides of glycoproteins: bioprocess factors
affecting oligosaccharide structure and their effect on
glycoprotein properties. Biotechnology 1991, 9 :1347-1355.
17.
Jenkins N, Parekh RB, James DC: Getting the glycosylation right:
implications for the biotechnology industry. Nat Biotechnol 1996,
14 :975-981.
18. Grabenhorst E, Schlenke P, Pohl S, Nimtz M, Conradt HS: Genetic
•
engineering of recombinant glycoproteins and the glycosylation
pathway in mammalian host cells. Glycoconjugate J 1999, 16:81-97.
This paper provides a very comprehensive discussion of the history and
scope of glycosylation issues that affect the recombinant protein production
field. It includes several examples of glycosylation engineering to introduce
or modify oligosaccharide structures on model proteins including sLEX
motifs and α2,6-linked sialic acid residues.
19. Weikert S, Papac D, Briggs J, Cowfer D, Tom S, Gawlitzek M,
•• Lofgren J, Mehta S, Chisholm V, Modi N et al.: Engineering Chinese
hamster ovary cells to maximize sialic acid content of
recombinant glycoproteins. Nat Biotechnol 1999, 17 :1116-1121.
This report contains several examples of engineering CHO cells to overexpress one or more glycosyltransferases and includes extensive glycan
characterization on materials produced in bioreactor production cultures.
20. Umaña P, Jean-Mairet J, Moudry R, Amstutz H, Bailey JE: Engineering
glycoforms of an antineuroblastoma IgG1 with optimized
antibody-dependent cellular cytotoxic activity. Nat Biotechnol
1999, 17 :176-180.
121
26. Fussenegger M, Schlatter S, Dätwyler D, Mazur X, Bailey JE:
Controlled proliferation by multigene metabolic engineering
enhances the productivity of Chinese hamster ovary cells. Nat
Biotechnol 1998, 16 :468-472.
27.
••
Kaufmann H, Mazur X, Marone R, Bailey JE, Fussenegger M:
Comparative analysis of two controlled proliferation strategies
regarding product quality, influence on tetracycline-regulated
gene expression, and productivity. Biotechnol Bioeng 2001,
72 :592-602.
This report compares p27 with temperature shift mediated proliferation
control on cellular productivity and product quality. The results suggest that
the effect of temperature shift on productivity may occur, at least in some
instances, through induction of cold-sensitive transcriptional regulators.
28. Koster M, Kirchhoff S, Schaper F, Hauser H: Proliferation control of
mammalian cells by the tumor suppressor IRF-1. Cytotechnology
1995, 18 :65-75.
29. Geserick C, Bonarius HPJ, Kongerslev L, Hauser H, Mueller PP:
Enhanced productivity during controlled proliferation of BHK cells
in continuously perfused bioreactors. Biotechnol Bioeng 2000,
69 :266-274.
30. Miller W M, Blanch HW, Wilke CR: A kinetic analysis of hybridoma
growth and metabolism in batch and continuous suspension
culture: effect of nutrient concentration, dilution rate and pH.
Biotechnol Bioeng 1988, 32 :947-965.
31. Hayter PM, Curling EMA, Gould ML, Baines AJ, Jenkins N, Salmon I,
Strange PG, Bull AT: The effect of dilution rate on CHO cell
physiology and recombinant interferon- γ production in glucoselimited chemostat culture. Biotechnol Bioeng 1993, 42 :1077-1085.
32. Lee FW, Elias CB, Todd P, Kompala DS: Engineering Chinese
hamster ovary (CHO) cells to achieve an inverse growthassociated production of a foreign protein, β-galactosidase.
Cytotechnology 1998, 28 :73-80.
33. Mastrangelo AJ, Hardwick JM, Zou S, Betenbaugh MJ: Part II.
Overexpression of Bcl-2 family members enhances survival of
mammalian cells in response to various culture insults. Biotechnol
Bioeng 2000, 67 :555-564.
34. Laken HA, Leonard MW : Understanding and modulating apoptosis
in industrial cell culture. Curr Opin Biotechnol 2001, 12 :175-179.
35. Kim NS, Lee G M: Overexpression of Bcl-2 inhibits sodium
butyrate-induced apoptosis in Chinese hamster ovary cells
resulting in enhanced humanized antibody production. Biotechnol
Bioeng 2001, 71 :184-193.
36. Figueroa B Jr, Sauerwald TM, Mastrangelo AJ, Hardwick JM,
•• Betenbaugh MJ: Comparison of Bcl-2 to a Bcl-2 deletion mutant
for mammalian cells exposed to culture insults. Biotechnol Bioeng
2001, 73 :211-222.
This study provides the very interesting observation that wild-type Bcl-2 is
degraded more rapidly than a mutant Bcl-2 lacking the loop domain. The study
also suggests that control of proteolytic processing of anti-apoptotic genes
following pro-apoptotic insults may provide a new avenue for cell-death control.
37.
Baker KN, Rendall MH, Hills AE, Hoare M, Freedman RB, James DC:
Metabolic control of recombinant protein N-glycan processing in
NS0 and CHO cells. Biotechnol Bioeng 2001, 73 :188-202.
21. Davies J, Jiang LY, Pan L-Z, LaBarre MJ, Anderson D, Reff M:
Expression of GnTIII in recombinant anti-CD20 CHO production
cell line: expression of antibodies with altered glycoforms leads
to an increase in ADCC through higher affinity for FcγRIII.
Biotechnol Bioeng 2001, 74 :288-294.
38. Hills AE, Patel A, Boyd P, James DC: Metabolic control of
recombinant monoclonal antibody N-glycosylation in GS-NS0
cells. Biotechnol Bioeng 2001, 75 :239-251.
22. Grabenhorst E, Hoffmann A, Nimtz M, Zettlmeissl G, Conradt HS:
Construction of stable BHK-21 cells coexpressing human
secretory glycoproteins and human Gal( β1–4)GlcNAc-R
α2,6-sialyltransferase. Eur J Biochem 1995, 232 :718-725.
39. De Zengotitia VM, Miller W M, Aunins JG, Zhou W : Phosphate
feeding improves high cell concentration NS0 myeloma culture
performance for monoclonal antibody production. Biotechnol
Bioeng 2000, 65 :566-576.
23. Monaco L, Marc A, Eon-Duval A, Acerbis G, Distefano G, Lamotte D,
Engasser J-E, Soria M, Jenkins N: Genetic engineering of
α2,6-sialyltransferase in recombinant CHO cells and its effects on
the sialylation of recombinant interferon- γ. Cytotechnology 1996,
22 :197-203.
40. Chang KH, Kim KS, Kim JH: N -Acetylcysteine increases the
biosynthesis of recombinant EPO in apoptotic Chinese hamster
ovary cells. Free Radic Res 1999, 30 :85-91.
24. Irani N, Wirth M, van den Heuvel J, Wagner R: Improvement of
primary metabolisms of cell cultures by introducing a new
cytoplasmic pyruvate carboxylase reaction. Biotechnol Bioeng
1999, 66 :238-246.
25. Chen K, Liu Q, Xie L, Sharp PA, Wang DIC: Engineering of a
mammalian cell line for reduction of lactate formation and high
monoclonal antibody production. Biotechnol Bioeng 2001,
72 :55-61.
41. Zanghi JA, Fussenegger M, Bailey JE: Serum protects protein-free
competent Chinese hamster ovary cells against apoptosis
induced by nutrient deprivation in batch culture. Biotechnol Bioeng
1999, 64 :108-119.
42. Balcarel RR, Stephanopoulos G: Rapamycin reduces hybridoma
cell death and enhances monoclonal antibody production.
Biotechnol Bioeng 2001, 76 :1-10.
43. Baneyx F: Recombinant protein expression in Escherichia coli.
Curr Opin Biotechnol 1999, 10 :411-421.
122
Biochemical engineering
44. Spiess C, Beil A, Ehrmann M: A temperature-driven switch from
chaperone to protease in a widely conserved heat shock protein.
Cell 1999, 97 :339-347.
62. Oh MK, Liao JC: DNA microarray detection of metabolic
responses to protein overproduction in Escherichia coli. Metabol
Eng 2000, 2 :201-209.
45. Lin W -J, Huang S-W, Chou CP: DegP-coexpression minimizes
inclusion-body formation upon overproduction of recombinant
penicillin amylase in Escherichia coli. Biotechnol Bioeng 2001,
73 :484-492.
63. Hoffman F, Rinas U: Kinetics of heat-shock response and inclusion
body formation during temperature-induced production of basic
fibroblast growth factor in high-cell-density cultures of
recombinant Escherichia coli. Biotechnol Prog 2000,
16 :1000-1007.
46. Bessette PH, Qiu J, Bardwell JCA, Swartz JR, Georgiou G: Effect of
sequences of the active-site dipeptides of DsbA and DsbC on
in vivo folding of multidisulfide proteins in Escherichia coli.
J Bacteriol 2001, 183 :980-988.
47.
Joly JC, Leung WS, Swartz JR: Overexpression of Escherichia coli
oxidoreductases increases recombinant insulin-like growth
factor-I accumulation. Proc Natl Acad Sci USA 1998,
95 :2773-2777.
48. Bothmann H, Plückthun A: The periplasmic Escherichia coli
peptidylprolyl cis,trans-isomerase FkpA. J Biol Chem 2000,
275 :17100-17105.
49. Levy R, Weiss R, Chen G, Iverson BL, Georgiou G: Production of
•• correctly folded Fab antibody fragment in the cytoplasm of
Escherichia coli trxB gor mutants via the coexpression of
molecular chaperones. Protein Expres Purif 2001, 23 :338-347.
This study demonstrates the potential for folding and assembling antibody
fragments in the cytoplasm, which provides an interesting alternative to
periplasmic approaches.
50. Thomas JG, Baneyx F: ClpB and HtpG facilitate de novo protein
folding in stressed Escherichia coli cells. Mol Microbiol 2000,
36 :1360-1370.
51. Smolke CD, Carrier TA, Keasling JD: Coordinated, differential
•• expression of two genes through directed mRNA cleavage and
stabilization by secondary structures. Appl Environ Microbiol 2000,
66 :5399-5405.
This approach represents a potentially powerful new technique for controlling the relative level of expression of different recombinant proteins.
52. Simmons LC, Yansura DG: Translational level is a critical factor for
the secretion of heterologous proteins in Escherichia coli. Nat
Biotechnol 1996, 14 :629-634.
53. Khlebnikov A, Risa O, Skaug T, Carrier TA, Keasling JD: Regulatable
arabinose-inducible gene expression system with consistent
control in all cells of a culture. J Bacteriol 2000, 182 :7029-7034.
54. Han SJ, Chang HN, Lee H: Characterization of an oxygendependent inducible promoter, the nar promoter of Escherichia
coli, to utilize in metabolic engineering. Biotechnol Bioeng 2001,
72 :573-576.
55. Wilms B, Hauck A, Reuss M, Syldatk C, Mattes R, Siemann M,
Altenbuchner J: High-cell-density fermentation for production of
L- N -carbamoylase using an expression system based on the
Escherichia coli rhaBAD promoter. Biotechnol Bioeng 2001,
73 :95-103.
56. Akesson M, Hagander P, Axelsson JP: Avoiding acetate
accumulation in Escherichia coli cultures using feedback control
of glucose feeding. Biotechnol Bioeng 2001, 73 :223-230.
57.
Zhu T, Phalakornkule C, Koepsel RR, Domach MM, Ataai MM: Cell
growth and by-product formation in a pyruvate kinase mutant of
E. coli. Biotechnol Prog 2001, 17 :624-628.
58. Andersson CIJ, Holmberg N, Farres J, Bailey JE, Bulow L, Kallio PT:
Error-prone PCR of Vitreoscilla hemoglobin ( VHb) to support the
growth of microaerobic Escherichia coli. Biotechnol Bioeng 2000,
70 :446-455.
59. Bollinger CJT, Bailey JE, Kallio PT: Novel hemoglobins to enhance
microaerobic growth and substrate utilization in Escherichia coli.
Biotechnol Prog 2001, 17 :798-808.
60. Aydin S, Webster DA, Stark BC: Nitrite inhibition of Vitreoscilla
hemoglobin (VHb) in recombinant E. coli: direct evidence that
VHb enhances recombinant protein production. Biotechnol Prog
2000, 16 :917-921.
61. Frey AD, Fiaux J, Szyperski T, Wüthrich K, Bailey JE, Kallio PT:
Dissection of central carbon metabolism of hemoglobinexpressing Escherichia coli by 13 C nuclear magnetic resonance
flux distribution analysis in microaerobic bioprocesses. Appl
Environ Microbiol 2001, 67 :680-687.
64. Gill RT, DeLisa MP, Valdes JJ, Bentley W E: Genomic analysis of
high-cell-density recombinant Escherichia coli fermentation and
“cell conditioning” for improved recombinant protein yield.
Biotechnol Bioeng 2001, 72 :85-95.
65. Richins R, Chen W : Effects of FIS overexpression on cell growth,
rRNA synthesis, and ribosome content in Escherichia coli.
Biotechnol Prog 2001, 17 :252-257.
66. DeLisa MP, Wu C-F, Wang L, Valdes JJ, Bentley W E: DNA
microarray-based identification of genes controlled by
autoinducer 2-stimulated quorum sensing in Escherichia coli.
J Bacteriol 2001, 183 :5239-5247.
67.
Andersen DC, Swartz J, Ryll T, Lin N, Snedecor B: Metabolic
oscillations in an E. coli fermentation. Biotechnol Bioeng 2001,
75 :212-218.
68. Weikert C, Canonaco F, Sauer U, Bailey JE: Co-overexpression of
RspAB improves recombinant protein production in Escherichia
coli. Metabol Eng 2000, 2 :293-299.
69. Browning ST, Shuler ML: Towards the development of a minimal
cell model by generalization of a model of Escherichia coli: use of
dimensionless rate parameters. Biotechnol Bioeng 2001,
76 :187-192.
70. Edwards JS, Ibarra RU, Palsson BO: In silico predictions of
Escherichia coli metabolic capabilities are consistent with
experimental data. Nat Biotechnol 2001, 19 :125-130.
71. Hoffman F, Posten C, Rinas U: Kinetic model of in vivo folding and
inclusion body formation in recombinant Escherichia coli.
Biotechnol Bioeng 2001, 72 :315-322.
72. Srivastava R, Peterson MS, Bentley W E: Stochastic kinetic analysis
of the Escherichia coli stress circuit using σ32-targeted antisense.
Biotechnol Bioeng 2001, 75 :120-129.
73. Giddings G, Allison G, Brooks D, Carter A: Transgenic plants as
factories for biopharmaceuticals. Nat Biotechnol 2000,
18 :1151-1155.
74. Doran P: Foreign protein production in plant tissue cultures.
Curr Opin Biotechnol 2000, 11 :199-204.
75. Larrick JW, Thomas DW : Producing proteins in transgenic plant
and animals. Curr Opin Biotechnol 2001, 12 :411-418.
76. Sharp JM, Doran PM: Characterization of monoclonal antibody
•• fragments produced by plant cells. Biotechnol Bioeng 2001,
73 :338-346.
This report identifies and analyzes the potential for proteolytic degradation of
antibodies produced in plant cells.
77.
Marchal I, Jarvis DL, Cacan R, Verbert A: Glycoproteins from insect
cells: sialylated or not? Biol Chem 2001, 382 :151-159.
78. Ailor E, Takahashi N, Tsukamoto Y, Masuda K, Rahman BA, Jarvis DL,
Lee YC, Betenbaugh MJ: N-Glycan patterns of human transferrin
produced in Trichoplusiani insect cells: effects of mammalian
glycosyltransferase. Glycobiology 2000, 10 :837-847.
79. Breitbach K, Jarvis DL: Improved glycosylation of a foreign protein
by Tn-5B1-4 cells engineered to express mammalian
glycosyltransferases. Biotechnol Bioeng 2001, 74 :230-239.
80. Jarvis DL, Howe D, Aumiller JJ: Novel baculovirus expression
•• vectors that provide sialylation of recombinant glycoproteins in
lepidopteran insect cells. J Virol 2001, 75 :6223-6227.
This study provides evidence for sialylation in insect cells transfected with
β1,4-galactosyltransferase and α2,6-sialyltransferase.
81. Joshi L, Shuler ML, Wood HA: Production of sialylated N-linked
glycoproteins in insect cells. Biotechnol Prog 2001, 17 :822-827.
82. Tomiya N, Ailor E, Lawrence SM, Betenbaugh MJ, Lee YC:
Determination of nucleotides involved in protein glycosylation by
high-performance anion-exchange chromatography: sugar
Recombinant protein expression for therapeutic applications Andersen and Krummen
nucleotide contents in cultured insect cells and mammalian cells.
Anal Biochem 2001, 293 :129-137.
83. Goodrick JC, Xu M, Finnegan R, Schilling BM, Schiavi S, Hoppe H,
Wan NC: High-level expression and stabilization of recombinant
human chitinase produced in a continuous constitutive Pichia
pastoris expression system. Biotechnol Bioeng 2001, 74 :492-497.
84. Wang Y, Liang Z-H, Zhang Y-S, Yao S-Y, Xu Y-G, Tang Y-H, Zhu S-Q,
Cui D-F, Feng Y-M: Human insulin from a precursor overexpressed
in the methylotrophic yeast Pichia pastoris and a simple
procedure for purifying the expression product. Biotechnol Bioeng
2001, 73 :74-79.
85. d’Anjou MC, Daugulis AJ: A rational approach to improving
productivity in recombinant Pichia pastoris fermentation.
Biotechnol Bioeng 2001, 72 :1-11.
86. Hellwig S, Emde F, Raven NPG, Henke M, van der Logt P, Fischer R:
Analysis of a single-chain antibody production in Pichia pastoris
using on-line methanol control in fed-batch and mixed-feed
fermentations. Biotechnol Bioeng 2001, 74 :344-352.
87.
Frykman S, Srienc F: Cell-cycle-dependent protein secretion by
Saccharomyces cerevisiae. Biotechnol Bioeng 2001, 76 :259-268.
88. Gorgens JF, van Zyl W H, Knoetze JH, Hahn-Hagerdal B: The
metabolic burden of the PGK1 and ADH2 promoter systems for
heterologous xylanase production by Saccharomyces cerevisiae
in defined medium. Biotechnol Bioeng 2001, 73 :238-245.
89. Kim D-M, Swartz JR: Regeneration of adenosine triphosphate from
glycolytic intermediates for cell-free protein synthesis. Biotechnol
Bioeng 2001, 74 :309-316.
123
90. Shimizu Y, Inoue A, Tomare Y, Suzuki T, Yokogawa T, Nishikawa K,
•• Ueda T: Cell-free translation reconstituted with purified
components. Nat Biotechnol 2001, 19 :751-755.
This report provides an interesting demonstration that protein translation can
be accomplished in a system consisting entirely of purified components. This
system could be useful both for studying the effects of various components
and for comparison with systems using cell extracts (see [89]).
91. Leung W -LS, Swartz JR: Process for bacterial production of
proteins. US Patent 6,180,367, 2001.
92. Leung W -LS, Swartz JR: Process for bacterial production of
polypeptides. US Patent 6,258,560, 2001.
93. Morita M, Asami K, Tanji Y, Unno H: Programmed Escherichia coli
cell lysis by expression of cloned T4 phage lysis genes.
Biotechnol Prog 2001, 17 :573-576.
94. Curvers S, Brixius P, Klauser T, Thommes J, Weuster-Botz D, Takors R,
•
Wandrey C: Human chymotrypsin B production with Pichia pastoris
by integrated development of fermentation and downstream
processing. Part 1. Fermentation. Biotechnol Prog 2001, 17:495-502.
This thorough analysis illustrates the integration of fermentation and recovery
decisions in process development.
95. Thommes J, Halfar M, Gieren H, Curvers S, Takors R, Brunschier R,
Kula M-R: Human chymotrypsin B production from Pichia pastoris
by integrated development of fermentation and downstream
processing. Part 2. Protein recovery. Biotechnol Prog 2001,
17 :503-512.
96. Caubin J, Martin H, Roa A, Cosano I, Pozuelo M, de la Fuente JM,
Sanchez-Puelles JM, Molina M, Nombela C: Choline-binding domain
as a novel affinity tag for purification of fusion proteins produced
in Pichia pastoris. Biotechnol Bioeng 2001, 74 :164-171.