Table Of Content1
Overview of Experimental Strategies
on the Detection and Isolation
of Recombinant Proteins and Their Applications
Rocky S. Tuan
1. Introduction
Recent advances m recombinant DNA technology have permitted the direct
cloning of DNA fragments (either derived from naturally occurring or artifi-
cially designed gene sequences) mto various cloning vectors mcludmg bacte-
riophages, plasmids, and viruses. Such recombinant constructs represent the
basic reagents of molecular biology. A major application utihzmg cloned DNA
sequences is the expression of the cloned DNA mto a protein product, i.e., the
expression of recombinant genes. Because the cloned DNA sequences may
be modified or altered, recombinant expression technology thus enables the
mvestigator to “custom-design” the final protem product Furthermore, most
expression vectors are designed to allow the linking of various “tags” to the
expressed recombinant protein to facilitate subsequent detection and isolation.
This chapter provides a brief overview of the technologies currently employed
m “taggmg” expressed recombinant protems and the corresponding detection
and isolation methodologies, as well as some of the applications utilizing
recombinant gene products.
2. Molecular Tags and Reporters
The basic strategy m “labeling” or “tagging” a cloned sequence is to place
either upstream or downstream a translationally m-frame sequence correspond-
ing to a polypeptide domain or protein that exhibits highly active or distmct
properties not found m the host cell. In this manner, the recombinant hybrid
protein, containing the tag and the desired expressed gene product, may be
detected and/or isolated on the basis of the unique properties of the tag. In
From Methods m Molecular Bology, vol 63 Recombrnant Rote/n Protocols
Detecftoon and /so/at/on Edlted by R Tuan Humana Press Inc , Totowa, NJ
3
4 Tuan
some instances, an additional sequence correspondmg to a specific protease
cleavage sate is Inserted between the tag and the cloned sequence, such that
treatment of the final recombmant hybrid protein with the appropriate protease
produces the desired gene product from the tag. Chapter 2 (Groskreutz and
Schenborn) m this book provides further background for the general rationale
used in constructmg an expression vector.
2. I. Enzymes
Owmg to their ability to catalyze specific reactions yielding distmct, detect-
able products, enzymes are probably the most popular molecular tag for
expression of recombmant genes. The most commonly used enzymes include.
chloramphenicol acetyltransferase (CAT), firefly luctferase; j3-galactosidase,
alkalme phosphatase; and P-glucuromdase Some of the key reasons for select-
mg these enzymes as functional labels include high signal-to-background ratios
of the catalyzed reactions, high stability of enzyme activity, and the high sensi-
tivity for detection A number of methods are currently m use for the detection
of enzyme activity, mcludmg standard calorimetric assays,m ore sensitive fluo-
rescence- or lummescence-based procedures, chromogenic histochemistry, and
immunohistochemistry or solution-phase immunoassays such as radtoim-
munoassay or enzyme-lmked immunosorbent assay (ELISA). The specific
characteristics of some of these enzymes and then- respective detection pro-
tocols are presented m detail m a number of chapters m this book (Chapters 3,
4, 5, and 6).
2.2. Ligand-Binding Labels
Another type of molecular interaction that has been exploited to generate
detectable activities m recombmant gene expression mcludes those mvolving
specific, high-affimty ligand binding. In this manner, the recombmant product
possessest he ability to interact with a specific hgand, which ideally is not a
property of the host cell proteins. Using a labeled ligand, the correspondmg
recombinant product may be clearly identified. Alternatively, another reagent,
either a protein or a chemical (which is itself labeled), may be used to detect
the bound ligand, and thus the recombmant protem. In many instances, the
ligand may be immobilized onto a solid support, such as chromatography
resins and gels, to develop affinity fractionation methods for isolation and
purification of the desired recombmant product. Examples of these proto-
cols may be found m a number of chapters m this book, dealing with spe-
cific ltgand-binding entitles such as: maltose-bmdmg protein, which allows
purification of the chimeric protein on amylose columns; Protein A, which
recognizes the Fc domain of mnnunoglobulin G; streptavtdm, which binds with
extremely high affinity and specificity to btotm, and hexahistidme peptide se-
Overview 5
quence, which has hrgh metal affinity, i.e., applicable for affimty purification
on nickel-mtrilotriacetate column. These topics are covered m detail m Chap-
ters 9, 10, 11, and 12
2.3. Expression-Coupled Gene Activation
Another means to detect recombinant gene expression, which has recently
gamed substantial popularity, is the couplmg of recombinant gene expression
to the transactivation of another unique gene This approach, an example of
which is the yeast two-hybrid system, is particularly useful for the detection of
interacting proteins, and the assay is performed m vivo rather than m vitro,
thus permittmg the detection of such proteins m their native, biologically active
state. The yeast two-hybrid system takes advantage of the fact that many
eukaryotic transcrtption activators are made up of structurally separable and
functionally independent domains. For example, the yeast transcriptional acti-
vator protein GAL4 contams a DNA-binding domain (DNA-BD), which rec-
ognizes a 17 base-pan DNA sequence, and an acttvation domain (AD). Upon
DNA-BD binding to the specific upstream region of GAL4-responsive genes,
the AD interacts with other components of the machinery to initiate transcrip-
tion. Thus, both domams are needed m an mteractive manner for specific gene
activation to take place. In the popular yeast two-hybrid system, the two GAL4
domains are separately fused to genes encoding proteins that interact with each
other, and these recombinant hybrid proteins are expressed in yeast. Interac-
tion of the two-hybrid proteins brings the two GAL4 domains m close enough
proximity to form a functional gene acttvator, resultmg m the expression of
specific reporter gene(s), thereby rendering the protein interaction, 1.e , expres-
sion of the desired recombinant protein, phenotypically identifiable. In prac-
tice, the target protein gene is ligated to the DNA-BD m the form of an
expression vector. The gene of interest, whose acttvtty Includes mteraction with
the target protein, IS ligated into an AD vector. The two hybrid plasmids are
then cotransformed mto specialized yeast reporter strain. Expression of the
desired gene thus activates a known GAL4 responsive gene(s) and confers spe-
cific phenotype to the host cell, which can be selectively identified Protocols
utilizing the two-hybrid system and its variants are described m several chap-
ters m this book (Chapter 12, 15, and 16).
2.4. lmmunospecific Detection
Another type of recombinant label or tag consists of components to which
specific antibodies are available In thts manner, mmmnoassays and nnmuno-
affinity chromatography may be used efficiently to detect and isolate, respec-
tively, the recombinant protem. Momand and Sepehrma (Chapter 14) illustrate
how this prmciple may be exploited using recombinant p53 as an example, and
6 Tuan
Olesen et al. (Chapter 7) present methodologres based on chemrlummescent
rmmunoassays using enzyme-conjugated secondary antibodies. Recombinant
protem expressing cells may also be cloned and detected by tmmunodetectron
methods as described by Gibson et al. (Chapter 8).
2.5. Expression-Coupled Alteration of Cellular Activity
The rdentificatton and clonmg of multrdrug resistance genes such as MDRl,
which is responsible for the simultaneous resistance of cells to multtple struc-
turally and functionally unrelated cytotoxtc agents, offers the potential to use
such genes m fusion gene constructs for the purpose of detection and isolatron
of the expressing cellular clone. Germann (Chapter 13) describes such an
application pertaining to P-glycoprotem.
2.6. Labels with Unique Chemical Characteristics
The green fluorescent protein (GFP) of the jellyfish, Aequora victona, fluo-
resces following the transfer of energy from the Ca2+-actrvated photoprotein,
aequorin. GFP has been cloned, and, when expressed in prokaryotic and
eukaryottc cells, yields green fluorescence when exctted by blue or ultravtolet
light. Recent developments have focused on utrlizing the GFP as a reporter gene
to permit the detection of recombinant gene expression m vlvo (see below).
3. Detection of Gene Expression
The expression of spectfic genes m a recombinant form consisting of cht-
merit or hybrid labels has greatly facrlitated then detection. A number of chap-
ters m this book (Chapters 18, 19, 20, 21, 22, 23, and 24) focus on recent
developments in the detection of gene expression zn situ, utilizing DNA or
RNA probes, as well as PCR amplificatron. Smce zns ztu hybridizatron, in sztu
PCR, and label-spectfic htstochemrstry (e.g., P-galactosrdase hlstochemrstry),
utrlizmg tissue sectron or whole-mount tissues and embryos, yield mformatron
on gene expression at the native, indrvtdual cell and tissue level, they are pow-
erful techniques in gammg mformation on the functtonal aspects of gene
expression. In particular, in transgemc expertmentatrons, where a transgene IS
introduced into and expressed in an animal, the abtlrty to correlate gene
expression and altered cell/tissue phenotype allows the investigator to directly
assess the function of the gene of interest. Examples of recombmant label-
specific detection protocols include those based on P-galactosidase (Chapter
18) and the fluorescent jellyfish GFP (Chapter 24). In situ hybridization IS
based on hybrrdrzatron to spectfic mRNA sequences by labeled DNA and RNA
probes (Chapters 19,20,2 1, and 22), whereas sequence-specific amplrficatton
by in situ PCR provides both gene expression detection and cloning possrbrlr-
ties (Chapter 23). By coupling immunohrstochemrstry with zn sztu hybridlza-
Overview 7
tion, it IS posstble to examme gene expression at both the protein and mRNA
levels (Chapters 19 and 22).
4. Applications of Recombinant Gene Expression
The productron of recombinant gene products IS one of the maJor success
stories of modern molecular biology. This book samples some of the applrca-
trons to Illustrate the present state and future potentral of such a technology.
Thus, m addmon to answering fundamental questions related to regulation of
gene expression, gene structure and function, and other basic issues of molecu-
lar biology, the technology of recombinant genes has revolutionized modern
brotechnology and bromedicme. For example, gene therapy, which arms to
compensate for gene defects and/or deliver therapeutrc gene products for spe-
c~fic diseases resultmg from defective genes, is crrtically and totally dependent
on the design of expression and delivery vectors, which permit targeted and
regulated expression of the cloned gene(s) (see Chapters 28, 29, 30, and 3 1).
On the other hand, recombinant gene technology also makes rt possible to cus-
tom-desrgn gene sequences to “blosyntheslze” novel bropolymers of unique
physrcochemical properties (see Chapter 27), which may be used for apphca-
trons m biomaterral, pharmaceutical, agricultural, and other mdustrres Finally,
it should be noted that the current state of recombmant technologtes IS capable
of utibzmg a wade spectrum of manufacturing umts (the “btoreactors”) for the
custom-designed protein products, mcludmg E. colz (Chapter 26), yeast, cul-
tured cells, and transgenic animals (Chapter 25 j.
2
Reporter Systems
Debyra Groskreutz and Elaine T. Schenborn
I. Introduction
Genetic reporter systems have contributed greatly to the study of eukaryotrc
gene expression and regulation. This chapter will descrtbe what an Ideal
reporter system IS and outline the many uses of genetlc reporters. Furthermore,
the currently avarlable reporter genes and assays ~111b e described in terms of
their specific applications and limitations.
Reporter genes are most frequently used as indicators of transcrrptional
activity m cells (1). TypIcally, the reporter gene or cDNA IS jomed to a pro-
moter sequence m an expression vector that IS transferred mto cells. Followmg
transfer, the cells are assayed for presence of the reporter by duectly measur-
mg the amount of reporter mRNA, the reporter protein itself, or the enzymatic
actlvrty of the reporter protein. The ideal genetic reporter ts not endogenously
expressed in the cell type of interest, and the ideal reporter assay system has
the characteristrcs of being sensitive, quantrtatrve, rapid, easy, reproducible,
and safe.
Reporter genes are used for both m vitro and m vwo applications (reviewed
in ref. 2). Reporter systems are used to study promoter and enhancer sequences,
trans-acting mediators such as transcriptton factors, mRNA processmg, and
translatron. Reporters are also used to monitor transfectron efficrenctes, pro-
tem-protein interactions, and recombmatlon events,
The E coEze nzyme, chloramphenicol acetyltransferase (CAT), was used in
the first pubhcatron descrrbmg genetic reporter vector and assay systems
desrgned for the analysis of transcriptional regulation in mammalian cells (3).
Since that time, several reporter genes and assays have been developed and
include P-galactosrdase, lucrferase, growth hormone (GH), P-glucuronidase
(GUS), alkaline phosphatase (AP), and most recently, green fluorescent pro-
From Methods m Molecular B/o/ogy, voi 63 Recombrnant Profern Protocols
Defectron and /so/at/on E&ted by R Tuan Humana Press Inc , Totowa, NJ
12 Groskreutz and Schenborn
ermmatlon codons
(for background,reductton)
Fig. 1. Plasmld DNA map of a generic reporter expression vector, pReporter, hlgh-
lighting regions of functional slgmficance Abbrevlatlons Amp’, amplclllm resistance
gene, fl on, orlgm of repllcatlon for filamentous phage; MCS, multiple clonmg sites,
poly (A), recogmtlon sequence for polyadenylatlon, orI, plasmld orlgm of rephcatlon
tem (GFP). These available genetlc reporters and their potential appllcatlons
are summarized in Section 4
2. Ideal Characteristics of Reporter Vectors
The design of a typical reporter expression vector is described m Sections
2 l .-2 5. and summarized m Fig. 1.
2.1. Vector Backbone Considerations
In addltlon to the components required m all expression vectors for optimal
protein productlon (see Chapter 25), there are addItIona conslderatlons for
deslgnmg reporter gene vectors First, the Ideal reporter vector contains no
regulatory binding sites or sequences other than the ones knowingly mserted
by the researcher. The presence of extraneous control elements can lead to
artlfactual results (4,5) such as increased or decreased background expression
of the reporter. Although attempts can be made to remove known bmdmg sites
from the reporter vectors and to reduce read-through transcrlptlon orlgmatmg
upstream of the reporter gene, the practical likelihood IS slim for removing all
potential regulatory sequences from several kllobasepalrs of DNA Therefore,
researchers usmg reporter vectors should be aware of this problem and use the
proper vector controls m their experiments.
Reporter plasmid vectors are generally propagated in E cob. Therefore, the
plasmid backbone contains an orlgm for DNA rephcatlon (on) and a gene for
selectlon, typically the ampicillin-resistance gene. Presence of an orlgm of rep-
Reporter Systems 13
hcation that allows a high copy number of plasmids m E toll (e.g., the pUC
on) facilitates large-scale DNA preparations of plasmid DNA. Presence of an
orrgm of replication for filamentous phage, such as the fl ori, allows for the
production of single-stranded DNA that IS useful for mutagenesis and sequenc-
mg applications.
2.2. Reporter Gene and Flanking Regions
The coding and flanking regions of reporter genes can be altered for both
improved performance and convenience. Altering naturally occurring target-
mg sequences of reporter proteins can be beneficial. Removal of the last 24
amino acids from the carboxyl end of alkaline phosphatase causes the reporter
enzyme to be secreted directly from the cells, avoidmg the need for cell lysis
(6). Additionally, elrmination of the peroxisomal targeting sequence of
Iuciferase allows luciferase to be transported to the cytosol rather than the
peroxisomes (7).
To maximize expression of the reporter gene, an optimal rrbosomal bmdmg
sequence (GCCGCCA/GCCATG...) (8) may be placed at the 5’ end of the
reporter gene. This sequence has been shown to increase the efficiency of trans-
lation and it also produces a convenient NcoI site, which can be used to create
N-terminal gene fusions with the reporter Removing regulatory binding sites
or altering cloning sites within reporter genes (7,9) can maximize performance
and convenience, respectively
2.3. Multiple Cloning Sites
Many reporter vectors contam two multiple clonmg sites (MCS). One is
located upstream of the reporter gene for cloning putative promoter or
enhancer/promoter regrons The other MCS is located elsewhere m the plas-
mtd for cloning regulatory elements, like enhancers, that act at a drstance.
Addmonal cloning sites can also be used to incorporate selectable gene mark-
ers for long-term expression of the reporter gene
Many convenience features may be incorporated mto an MCS. For example,
the presence of a cleavage site for a restriction enzyme that generates blunt
ends is useful because this allows any blunt-ended fragment to be inserted into
the MCS. The MCS can be designed to also include a sequence cleaved by
restrtction enzymes that generate 3’ overhangmg ends at one or both ends of
the MCS. A 3’ overhanging end provides the opportumty to perform nested
deletion analysrs with exonuclease III (IO).
The nucleotide sequences of an MCS located 5’ of the reporter gene should
be designed to avoid potential hairpin loops and upstream ATG sequences that
become part of the reporter mRNA. Either of these factors can decrease the
efficiency of translation of the reporter message (1 Z-1 7).
74 Groskreutz and Schenborn
2.4. Polyadenylation Signals
Polyadenylatton has been shown to enhance mRNA stability and translation
in mammalian cells (J8,19). Immediately following the reporter gene is a
polyadenylation (poly A) sequence that signals the addition of 200-250 adeny-
late residues to the 3’-end of an RNA transcript (20). Commonly used poly A
sequences in reporter vectors are derived from the SV40 early and late genes,
or the bovine growth hormone gene. For optimal expression, the SV40 late and
the bovine growth hormone poly A sites have been shown to be five-, and
threefold (21,22) more efficient than the SV40 early poly A at generating high
levels of steady-state mRNA.
A poly A signal inserted 5’ of the transcription unit can lower levels of gene
expression originating from cryptic promoter sequences in the vector back-
bone. Eltmmation of this background expression effectively increases the sen-
sttivtty of the reporter system (23). However, when mcorporatmg two poly A
signals within the same vector, nonhomologous regions contammg poly A sig-
nals should be used to reduce the chances of recombmation within the same
vector. A further reduction m background reporter expression from spurious
transcription within the vector backbone may be achieved at the translational
level by incorporatmg stop codons in all three reading frames upstream of the
reporter transcription unit.
2.5. lntron Effects on Reporter Gene Expression
The presence of an mtron m the mRNA sequence has been shown to increase
the level of expressed protein for particular cDNAs transfected mto mamma-
ban cells (24-27). Many reporter genes are derived from bacterial genes con-
tainmg no mtrons and, therefore, mtrons were added to vectors so that the
reporter genes more closely resembled the pattern of exons and mtrons m mam-
maltan genes. Early reporter vectors included the SV40 small-t antigen intron
3’ of the reporter gene for the purpose of increasing message stability and pro-
tein expression. More recently, however, it has been demonstrated that the
small-t intron m this posttion can lead to cryptic splicmg of mRNA from sites
within the reporter gene. Iromcally, the presence of this intron reduces protein
expression of CAT and @galactosidase by 1O -fold compared to the expresston
levels from the identical vectors lacking an mtron (26,281.
The effect of an mtron may need to be determined empirically because it is
dependent upon the specific gene sequence with which it is associated, and can
be different for in vitro and in vivo applications. For transient transfection stud-
ies, a 5’ intron increased luctferase expression three- to fivefold, although the
same mtron increased CAT expression over 20-fold (29). Studies with
transgemc animals have demonstrated that introns are generally required for
