Table Of ContentRecombinant Gene Expression Protocols
Overview of Experimental Strategies
for the Expression of Recombinant Genes
Rocky S. Tuan
1. Introduction
The direct clonmg of DNA fragments, etther dertved from naturally occurrmg
or artrficrally designed gene sequences, into various clomng vectors, mcludmg
bacteriophages, plasmids, and vu-uses, IS the cornerstone of modern molecular
biology. Such recombinant constructs represent the basic reagents of molecu-
lar biology One of the maJor apphcatlons utrhzmg cloned DNA sequences IS
the expression of the cloned DNA mto a protein product, 1.e , the expression of
recombmant genes Because the cloned DNA sequences can be modified or
altered, recombinant expression technology thus enables the mvestlgator to
custom-design the final protein product to sun specific requirements The pro-
duction of such recombinant gene products IS m fact one of the major success
stories of modern molecular biology. Thus, m addmon to answering funda-
mental questions related to regulation of gene expresston, gene structure and
function, and other basic issues of molecular biology, the technology of
recombinant genes has revolutlomzed modern brotechnology and blomedtcme
Applications m gene therapy, btopharmaceutics, bropolymers, and the like,
have major impacts on science, medlcme, agriculture, and mdustry
A major technologrcal requn-ement m successful expression of recombmant
genes 1s the choice of the proper vector/host system and the use of efficient
means to Introduce the recombmant gene construct into the host cells. This
chapter provides a brief overview of the technologtes addressing these aspects
as described m this book.
2. Escherichia co/i
The bacterium Eschenchza colz 1s undoubtedly the best understood organ-
ism m nature The extensive genetic and brochemtcal studies of E. colz during
From Methods m Molecular B/o/ogy, vol 62 Recombmant Gene Expression Protocols
Edlted by R Tuan Humana Press Inc , Totowa, NJ
3
the 1960s and 1970s is the foundation for modern recombmant DNA technol-
ogy Because E co11 1s easy to mampulate experimentally and grows rapidly m
relatively simple medmm, tt 1s the most commonly used host organism for
recombinant gene expression. Clonmg vectors used for E colz, commonly plas-
mtds, generally have the followmg charactertstics
1 A selectable marker to ensure mamtenance of the vector m the cell,
2 A regulatable transcnptronal promoter, such as lac, trp, and so on, that can be induced
to produce large quantities of mRNA transcripts from the cloned DNA sequence,
3 Appropriate translatlonal control sequences for rlbosome bmdmg and mltlatlon
of translation, and
4 Polylmker clonmg site to facilitate msertlon of the sequence of Interest mto
the vector
Several chapters m this book (Chapters 2,6,7, and 9) present in-depth mfor-
matton on the destgn of plasmtd vectors and promoters For protem expres-
sion, the recombmant construct needs to be mtroduced mto the appropriate
E colz host stram by transformanon Chapter 3 m this book addresses the design
of bacterial hosts for &-based expresston vectors. Another commonly used
expression vector for E colz 1s the bacteriophage h, which IS covered m Chap-
ter 4 Other important aspects of recombmant gene expresston m E colz are
covered m Chapter 5 (Strategies of Gene Fusion) and Chapter 8 (Expression
and Secretion of Proteins m E colz). Cell-free transcription and translation can
also be coupled for efficient gene expresston (Chapter 10).
3. Yeast
The baker’s yeast, Saccharomyces cerevzszae, IS among one of the stmplest
eukaryotes In addttton to having been characterized extensively m terms of its
genetics, yeast can be grown eastly either m the laboratory or m large-scale
fermentation cultures. For the purpose of expressing recombinant genes, the
yeast genome has been manipulated to reduce proteolysis as well as to ensure
proper posttranslational modifications For these reasons, S cerevzszae IS a pow-
erful and commonly used host orgamsm for the production of heterologous
recombinant proteins. Chapters m this book cover the followmg man-r topics
1 Chapter 11 provides an overvlew of the characterlstxs of yeast cloning vectors,
2 Chapters 12 and 13 describe two hrghly useful mducible expression cassettes,
utlhzmg the GAL4 and ADH promoters, respectively, and
3. Chapter 14 addresses the specifics of using a highly efficient constrtutrve expres-
sion vector containing the phosphoglycerate kmase (PGK) promoter
4. Viral Expression Vectors
As carriers of genetic mformation, viruses offer high mfecttvity, broad host
range, and the capacity to accommodate large gene fragments Viral vectors,
Overvlew 5
such as adenovirus and retrovirus, are currently being utilized actively m
destgnmg gene therapies Chapters m thts book provide background and tech-
nical mformation on several viral vectors currently m popular use for recombi-
nant gene expression, rncludmg retrovnuses (Chapter 17) vaccmla vn-uses
(Chapters 15 and 16), defective herpes vnus (Chapter 1 S), as well as baculo-
vnus for Insect cells (Chapter 19).
5. Eukaryotic Expression: Nonmammalian Systems
A popular eukaryotic expression system is the use of baculovn-uses m insect
cells (Chapter 19). Thts system allows posttranslattonal moditicatlon, process-
mg, and transport of the expressed proteins as m most higher eukaryotic cells.
In addition, the baculovtral expression system utilizes a helper-independent
vn-us which may be propagated to high titers m insect cells adapted to growth
m suspension cultures; thereby facilitating the tsolation of expressed proteins
m large quantities, since the majority of such protems remam soluble m insect
cells. The system also has the advantage that baculovnuses are nonmfectious to
vertebrate cells, and that their promoters are macttve m mammahan cells, which
are important considerations when oncogenes or potentially toxic proteins are to
be expressed. Generally, the level of expressed recombmant proteins m Insect
cells can reach up to half of the total cellular protein late m the mfecttous cycle,
greatly enhancing the production capability of the baculoviral system.
The seminal work of Gurdon and Brown, who injected various types of
RNAs mto both fertilized and unfertilized Xenopus eggs, was a major techno-
logical breakthrough m the molecular biology of development Such an
approach has served well as an experimental system to assay for gene active-
ties and functions. In this book, Chapters 20 and 21 present detailed back-
ground and techmcal mformation on the expression of heterologous or
exogenous genes m Xenopus oocytes, eggs, and embryos, including specific
blastomeres These methodologies have allowed many mvestigators to probe
the functtonal aspects of putatively developmentally important genes, m par-
ticular those involved m pattern formation and morphogenesis
6. Expression in Plants
The agricultural importance of expressing recombmant genes in plants can-
not be overestimated. In addition to enhancing crop yield, recent emphasis has
been on using plants as bioreactors for the production of bioactive reagents
Introduction of exogenous DNA mto plant cells requires overcommg the bar-
rier imposed by the cell wall. Detailed technical information is mcluded m this
book on recent developments m the mtroductron of exogenous genes mto plants
by electroporation (Chapter 34), microprojectile bombardment (Chapter 35),
the transforming bacterium Agrobactevium tumefaczens (Chapter 36), and the
6 Tuan
transformatton of mrcroalgae using silicon carbide whtskers (Chapter 37).
Among these, the btohsttc (bombardment) delivery system has also found
apphcatron m transgemc animal research as well as human gene therapy.
7. Expression in Mammalian Cells
Expression of foreign genes m mammahan cells probably represents the bulk
of current applrcattons m recombinant gene expression work, whtch has
become mcreasmgly cructal for the study of basic btologtcal questrons as well
as a means for productton of recombinant gene products for pharmaceuttcal,
agrtcultural, or other industrial uses The dtstmct advantages of expressing
recombinant genes m mammalian cells include the followmg reasons Cloned
DNA sequences derived from higher eukaryotes, erther m the form of cDNAs
or genomtc fragments, are readily and properly expressed because the full com-
pendium of posttranscrtpttonal acttvmes are functtonal m the host cell. In a
similar manner, the mammalian host cell IS also capable of posttranslattonal
modifications essential for the actlvatton and/or processmg of the translated
product, e.g., glycosylatton, subunit assembly, and so on. With the proper
selection of the host cell type, deltvery or compartmentahzatton of the final
gene product can be opttmtzed, thus allowing, for example, the secretion of
recombinant protein mto the culture medmm for efficient subsequent lsolatton
and purtficatton
The expression of recombinant genes m mammahan cells 1s covered m depth
m thts book, and addresses several aspects of this appllcatron*
1 The design of expressron vectors,
2 The means to mtroduce the user-designed expression construct Into the mam-
mahan host cells,
3 The ldenttficatton and selection of the transfected cells; and
4 Opttmlzatton of growth and recombmant gene expression of the transfected cells
Chapter 22 presents the background for vector destgn and suggesttons for
expresston strategies Several commonly used methods for efficient DNA
transfectton of mammahan cells are presented m this book. The classic and
wrdely used methods of calcmm phosphate coprectpttatton and DEAE-dex-
tran-mediated DNA transfectton are discussed m detail m Chapter 23. The use
of a pulse of hrgh electric field to transiently permeablltze cell membrane and
to allow the entry of macromolecules from the external medium mto the cell IS
known as electroporatton, and has recently gamed much populartty m DNA
transfectton. The theory and practice of this technology IS the subject of Chap-
ter 24 The catromc detergent, polybrene, 1s a relattvely inexpensive reagent
which has been recently shown to be highly efficient m medlatmg DNA trans-
fection of mammahan cells (Chapter 25).
Overview 7
Since DNA transfectton IS mcomplete and effictenctes range from less than
1% to, at best, 50-60%, the selection and isolation of transfected cells from
untransfected cells is crucial for the long-term propagation and homogeneity
of the cell populatton. Probably the most commonly used method IS the
cotransfection of a gene, ammoglycostde phosphotransferase, thus rendermg
the transfected cell resistant to the anttbrotrc neomycin, which 1sn ormally toxic
to mammahan cells by blocking ribosomal function and protein synthesis
In thus manner, by selectmg for cells that remain vrable m otherwise toxrc con-
centrations of the anttbtottc G418, clones of cells stably transfected wrth the
desired gene sequence can be Isolated. Although htghly useful, the G4 18 selec-
non protocol IS trme-consummg and works most efficiently only with rapidly
dividing cells, and 1s thus not applicable for transient expression studies. The
experimental prmctple of the procedure of magnetic affimty cell sorting (Chap-
ter 26) mvolves the cotransfectton of a gene sequence whtch encodes a cell
surface moiety, which may be subsequently recognized and tagged by specific
antibodies; m this manner, by applying magnetically tagged mnnunoselectton
technology, the transfected cells expressing the recombinant genes may be tso-
lated shortly after transfection (as short as 12-l 8 hours). Another hmttatton of
the G418 selection scheme IS that the neomycm-resistance gene IS not
amplifiable, and therefore does not promote overexpresston of the desired
recombinant gene. The use of the human multidrug resrstance (MDRI) gene IS
described m Chapter 27 as a selectable marker for gene transfection, drug
selectton, and gene amphficatron MDRI encodes a 170 ktlodalton glycopro-
tem, P-glycoprotem (Pgp), whrch functrons as a drug transporter to prevent the
mtracellular accumulatton of cytotoxtc agents and thereby confers resistance
to those agents. Colchrcme IS one of the drugs transported by Pgp, and expres-
sion of MDRZ cDNA confers colchtcme resistance to the cells. When the cells
are cultured m increasing concentrattons of colchtcme, the expression and copy
number of transfected A4DRl can be amplified, with concomttant ampllfica-
tron and overexpresston of the cotransfected, desired foreign gene.
The overall strategy of working wrth transfected cells to optrmtze their
growth, viability, and specific producttvtty for expression of recombinant pro-
teins, is the subject of Chapter 28 Methods covered include cell cloning,
suspension adaptation, adapting cells to reduced serum growth medmm, optt-
mizing medmm formulation, and momtormg of cell viability.
8. Expression in Transgenic Organisms
The advent of animal transgenests IS one of the modern technical wonders
of molecular biology Transgemc animals, expressing specific exogenous genes
or transgenes, have become an increasingly important system to examme and
analyze the brologtcal function(s) of the gene of interest. This book (Chapters
8 Tuan
29-33) presents the fundamental experimental prmclples m generating a num-
ber of widely studied species of transgemc animals These species include the
frultfly Drosophzla (Chapter 29), the nematodes Caenorhabhtis elegans and
Heterorhabdztzs bacterzophora (Chapter 30), mammals (mouse) (Chapter 3 l),
and avlan species (chlcken) (Chapter 32 and 33) These chapters provide the
readers with wide-rangmg, contemporary approaches to generatmg and using
such transgemc ammals for gene expresslon studies and apphcatlons.
2
Designing Expression Plasmid Vectors in E. co/i
Paulina Balbh
1. Introduction
The production of proteins IS one of the mam apphcatlons of genetic engi-
neering m biotechnology Even though standard cloning procedures are now
routine and a large variety of host-vector systems for gene expression are avail-
able, difficulties are encountered when theoretical strategiesa re put mto practice,
so gene expression 1ss till quite empmcal. E colzr emains an important host system
for the mdustrlal production of proteins from cloned genes, and considerable lore
has accumulated since the ploneermg gene expression expenments. The extensive
knowledge about E colz ‘s physiology and genetics accounts for its preferential
use as a host for gene expression. The inability of this organism to exert
certain posttranslational modifications of proteins that lead to correct folding
and activity represents its major drawback as a production organism.
The final amount of accumulated product in a fermentation process of a
genetically modified E co/z strain 1sd ependent upon the rates of biosynthesis
vs the rates of degradation, both of the final product (the target protein) and the
template (mRNA). Therefore, the two mam concerns when designing an
expression system should be the production of high levels of proteins and
mRNA, and the stablhty of both macromolecules. With this simple rule m
mind, there are four important components of an expression system that deter-
mme the final amount of accumulated protein:
1. The genetlcs of the expression system, such as the nucleotlde sequence and structure
of the target gene, the specific transcription and translation signals that direct the syn-
thesis of high amounts of protem and its mRNA, and the nature of the gene product
2 In the case of plasmld-based systems, a suitable vector with an adequate copy
number control mechanism, stability, a selective method, and a known replica-
tion mode IS essential
From Methods m Molecular B/o/ogy, vol 62 Recombmant Gene ExpressIon Protocols
Edited by R Tuan Humana Press Inc , Totowa, NJ
11
3 An appropriate host stram contalnlng the precise genetic traits necessary to work
in conJunctlon with the expresslon signals of the system, and/or to Increase
mRNA or protein stablllty
4 A proper cultlvatlon process, m which fermentation condltlons as well as the
mductlon procedure can be very important for the final yield of recombmant pro-
teins present m the culture at the time of harvestmg
These four elements should be carefully evaluated and must all fit mto a
processmg strategy, which should include not only plans for efficient produc-
tion, but a system for product recovery and purlficatlon The success or failure
of a process often depends on the mltlal choice of the host organism and the
expression system Whatever mformatlon 1s available on expression vectors,
fermentation and mductlon condltlons, nucleotlde sequences, and so forth, IS
vital for decision making This chapter presents practical recommendations for
designing strategies for gene expression or lmprovmg existing systems, and
emphaslzmg those particular aspects that can be Improved for higher product
output. Detailed dlscusslon of some particular aspects of gene expression m
plasmld-based systems has been reviewed elsewhere (1) For practical tech-
niques, it IS suggested that the reader consult the various books and labora-
tory manuals available that offer step-by-step mstructlons for DNA clonmg
and editing, or that hst an assortment of expression vectors with maps, descnp-
tlons, and uses (2-4)
2. Experimental Considerations
2.1. High-Level Synthesis of mRNA
Transcription mltlatlon xs the rate-llmltmg step for mRNA synthesis. In
E colz, the frequency of transcnptlon 1s regulated by a promoter sequence,
which can be modulated by a variety of mechanisms, namely the mterac-
tlon of one or more effector molecules with specific sequences m the
vlcmlty of the promoter Consequently, maxlmlzmg the efficiency of
transcrlptlon lnltlatlon with the mtroduction of a strong promoter IS the
key step necessary to attam the goal of high-level mRNA synthesis No
mRNA synthesis during the early stages of fermentation 1s desirable to
avoid lower growth rates ot lethality, therefore tightly regulated promoters
and strategies to completely evade transcrlptlon have been developed. Tran-
scription mltlatlon 1s often controlled by one of the followmg manners
temperature shift (3L pL and pR), chemical mductlon (lac and hybrid pro-
moters containing Its -10 region) or metabolic response @hoA, trp) There-
fore, the choice of promoter must take mto conslderatlon the other three
components of the system., namely strain genotype, fermentation condl-
tlons, and type of plasmld (1,5).
Express/on Pk~~rnid Vectors in E. co11 13
The occurrence of nonspecific transcription termination can affect the lev-
els of mRNA. This phenomenon is bound to be found when heterologous genes
are expressed, and analysis of the mRNA sequence often reveals the presence
of fortuitous mRNA-like termmator sequences. Disruption of these sequences
or addition of antitermmator sequences in the Y-end of the mRNA may solve
this problem. Finally, the transcriptional isolation of the genetic mformation
that leads to protein synthesis is important in order to avoid interference with
plasmid or cellular functions. Clomng of a strong transcription termmator at
the 3’-end of the gene m question often renders stability increase (1,5,6).
2.2. Enhancing the Stability of mRNA
Degradation of mRNA serves as an adaptative mechanism of cells to respond
rapidly to environmental changes, thus serving an important physiological role
for the modulation of gene expression Polynucleotide phosphorylase, RNases,
the presence or absence of ribosomes, the translation rate, secondary structures
present m the mRNA, the cell growth rate, and other factors have been shown
to influence mRNA decay. For high-level expression purposes, two simple
approaches have been used successfully the introduction of mRNA stabihzmg
5’-end enhancing sequences and the modulation of growth rates (1,5,6,8).
2.3. High-Level Protein Biosynthesis
Translation is the mRNA-directed process by which ammo acids are
assembled mto a polypeptide chain. Therefore, the mRNA contains the ele-
ments that condition :he initiation of translation: an urination AUG codon, a
Shine-Dalgarno sequence or ribosome binding site (RBS), and other ribonucle-
otides that confer folding and spacing attributes to mRNA (I, 6). The efficiency
of translation strongly depends on the sequence and structure of the RBS ele-
ment. At least four nucleotides of the general consensus AGGAGG should be
present, and the optimal spacing between the RBS and start codon should be
about nine nucleotides The mitiation codon AUG is preferred over GUG and
UUG. When the transcription initiation site is located far upstream of the trans-
lation mitiation site, upstream, out-of-frame inmation can occur that may
interfere with translation from the proper start. It is therefore useful m such
cases to include stop codons m all frames upstream of the RBS sequence (4).
2.4. Protein Stability
It is still not clear what determinants on a protein, either homologous or
heterologous, make it stable or labile when synthesized m E colz. Proteolysis
is a highly regulated process that exerts a global impact on cellular functions,
and although efforts to dimmish degradation have been focused on producing
strains with decreased capacity for proteolysis, no mutants completely defec-
tive on proteolysis have been obtamed. Suggested strain genotypes include
ion, ptr, ompT htrA, rpoH, and multiple mutants (5). A preferred strategy to
overcome selective degradation of proteins has been the generation of gene
fusions (translational fusions) that direct the synthesis of hybrtd protems. How-
ever, these hybrids require a cleavage process for the release of the target pro-
tem from the carrier Rapidly synthesized abnormal proteins often accumulate
as light-refractile aggregates known as mclusion bodies. The extent of mtrac-
ellular protein aggregates depends not only on the protein itself, but also on the
E. colz strain, the rate of protein synthesis and the growth conditions If the
protein of interest can be refolded adequately so that activity can be obtained,
the formation of mclusion bodies is desirable because proteolysis is lowered
and biosynthesis and purification might be facthtated. When the protein cannot
be refolded from mclusion bodies, it must be produced in soluble form, which
may not be easy to achieve in E colz. If this is the case, expression m a differ-
ent host may provide a more favorable environment for correct folding (68).
2.5. Choice of Expression Vector
The genetic material of the expression system can be maintained m E colz
either extrachromosomally on replicative plasmids, or integrated mto the host
genome. For years, the first approach has been preferred, although the second
is advantageous with respect to stabiltty and undesired copy number effects.
There is a considerable amount of mformatton m the literature concermng plas-
mid design, types, construction, structure, function, stability, and so forth, and
this mformation will not be presented here (Z-6). However, it IS Important to
emphasize the fact that rephcative plasmtds containing the DNA for gene
expression work rn conJunction with the host strain and the growth condmons
Plasmtd mstabihty of a recombinant plasmid m a fermentation is the mam cause
for reduction of the overall levels of accumulated product, which m turn
increases the production costs since the growth substrates are consumed by
nonproducttve cells The segregational mstabihty of a plasmid may be affected
by the overall genetic background of the host, but also by the copy number
of the plasmid, a high growth rate of the culture, and the genes contained m
the plasmid. These elements have to be studied separately m case of low prod-
uct output (9)
Plasmtd mstability is usually an msidious problem in overexpression sys-
tems. Integration of the mformation for gene expression mto the chromosome
has proven to be an excellent alternative method to tackle plasmid mstability
problems. Integration strategies include double- and single-crossover recom-
bination between homologous DNA fragments, transposon msertton, and phage
delivery systems. Lower copy number of the target gene is the only drawback
so far encountered (4)
