Information Panels 15.55 EXPRESSION OF CLONED GENES — Expression of cloned genes is perhaps the list area of molecular cloning tha retains some vestige ofthe pio- neering spirit. Because the routes for isolating genes and cDNAS are now so well mapped and because enzymes and reagents of high quality are available at almost every step of the way, cloners can usually trav- el from protein to gene in comfort and with reasonable confidence. However there is one region of molec tar cloning — expression of cloned genes —of which travellers must beware. Iisa maze of mirages where success depends on chance and experience may be of litle benef. It isthe one area in molecular cloning where investigators can do everything perfectly and stl se the experiments ‘The reasons for this are cleat Too litle is known about the mechanism of folding of proteins in fer ent organisms to predict which host-vector sytem might be best for a given protein, and few methods are available to increas the efficiency of folding oto prevent the aggregation, denaturation, andor degrada tion of foreign proteins expressed in enviconments that aze unnatural to them, Inthe absence of any useful ‘uiding principles, the expression of every CDNA or gene presents unique st of problems, which must be solved empirically Selection of an Expression System Five major expression systems have been developed to a point where they are reasonably portable from one laboratory to another, where the necessary components and vectors are readily available from commercial sources or public repositories, and where success can he documented with more than a hand of proteins, ‘The ost cells for these systems are E.coli, Bacillus subtilis, Saccharomyces cerevisiae, cultured insect cells and cultured mammalian cells. Very few, if any, laboratories have all five of these systems running and most investigators will ave to choose among the various options. Expression in E.coli. Pease see Chapter 15. + Expression in mammalian systems, Pease see Chapter 17. ‘» Expression in Saccharomyces cerevisiae. Although the range of available expression vectors is extensive (Emr 1990) few foreign proteins have been produced in yeas at levels that are more than modest. .cere= visiaeis generally a fairly inefficient factory for recombinant proteins. However, the extremely powerful genetic systems of the organism have been exploited in two important ways, neither of which requires 'massive overproduction of foreign proteins: First, because many mutants of S cerevisiae can be comple- ‘mented by expression of the homologous mammalian proteins, yeasts may be used as hosts for the iso- lation of mammalian cDNAs by complementation. Second, the powerful genetics of yeast have been ele- gantly exploited to develop selective systems forthe isolation of genes that encode pairs of interacting proteins (please see Chapter 18, Protocol |) ‘© Expression in Bacillus subtilis. 8. subtilis has a well-developed secretory system, and recombinant pro- teins can often be delivered to the medium in high yield ina soluble and active form. However, this i, not necessarily a great advantage since B subriis also secretes a number of proteases with powerful degradative activities. In addition, the range of vectors is rather limited, and there are few examples of proteins that can be expressed more efficiently in B. subtilis than in E. co. ‘© Expression in cultured insect cells, The baculovirus expression system is used to generate large quanti- ties of recombinant proteins in cultured insect cells. A potential advantage of the system i that proteins secreted from insect cells undergo a form of complex glycosylation. However, a major disadvantage is that expression ofthe foreign protein occurs during acute lytic infection of cells with a recombinant bac ulovirus stock. Protein production therefore occurs for only a short period of time and results in cell death. Consequently, there is a constant need to generate new virus stocks and new cells fr infection, Please se the information pane! on BACULOVIRUSES AND BACULOVIRUS EXPRESSION SYSTEMS in Chapter 17.15.56 Chapter 15: Expression of Cloned Genes in Escherichia coli E_ COLI EXPRESSION SYSTEMS Expression of foreign genes in bacteria has always been the Grail of molecular cloning, The eaely clonets ‘were remarkably confident that E, coli could be reprogrammed to elaborate any desired protein in quanti ties sufficient for any desired purpose, During the Great Recombinant DNA debate of the early 1970, the idea that there might be problems in constructing strains of E.coli that would make proteins to order was hardly questioned. Infact, much of the discussion centered around the degrees of physical and biological containment that would be required to protect the world from bacteria that expressed this or that foreiga gene. Synthesis of foreign proteins in prokaryotes was feared asthe approaching apocalypse by the Luddite fringe and was simultaneously peddled to Wall Steet as an apotheosis by the more entrepreneurial of the molecular biologists. As it turned out, neither the dark paranoia nor the day-glo optimism of those times was justified. The doomsayers were spectacularly wrong, as they since have been about other perceived dangers in molecular cloning. Inthe 20 years since the Asilomar conference, many thousands of stains of E, coli have been com structed that express foreign proteins, without untoward consequences. In recognition of this fact, most of the regulations that restricted expression work in E col were dropped in the United States a few years after Asilomar, with only token opposition from the hard-core pessimists. As forthe entrepreneurs, only a few of them became tich from proteins that were synthesized in E.coli For the most part, these proteins were small cytokines and growth factors whose secretion into medium could be easily engineered. Larger secreted pro- {cins and cytosolic proteins of al sizes proved to be far more difficult to produce After several years of effort, to solve problems of prokaryotic expression, most genetic engineering companies abandoned E.coli in favor of eukaryotic cells as the means to produce larger secreted proteins Although the solutions remain elusive, the reasons for these problems are clear enough. Too litle i known about the mechanism of folding of proteins in E, cal, and few infallible methods a to increase the efficiency of folding or to prevent the aggregation, denaturation, and/or degradation of for ign proteins expressed at high levels in an environment that is unnatural to them, In some cases, the use of ‘mutants of E.coli (eg, lon mutants) that are defective in degradation has proven helpful; in many other cases, solubility problems have been alleviated by expressing the polypeptide of interest as part ofa hybrid protein, However, most nonsecreted proteins synthesized in E. coli accumulate as denatured aggregates, called inclusion bodies, which can be purified by diferemtial centrifugation (lor review, please see Marston 1986; Cousens etal. 1987). These aggregates are insoluble in aqueous buffers at neutral pH, but they usual ly can be dissolved in butfers that are markedly acidic or alkaline or contain high concentrations of deter gents, organic solvents, or denaturants In a few case, it has then been possible to fold the solubilized pro- tein in vitro into an active or native state (or reviews, please see Marston 1986; Kohno et al. 1990), ‘Most success with E coli expression systems has been achieved with smal secretory proteins (Goeddel 1990), which can be translocated into the periplasmic space of E.coli from where they can be recovered and purified, sometimes in an active form. To achieve maximum efficiency, however, it is usually necessary 0 replace the eukaryotic hydrophobic signal sequences with a bacterial signal sequence and cleavage site. It is unreasonable to expect secretory proteins that depend on glycosylation or specific proteolytic cleavage to be recovered in an active state As indicated above, small cytosolic proteins and polypeptides (<100 residues in length) are best expressed in E.coli as fusion proteins. The carrier sequences often stabilize the protein of interest against svailabe cither intracellular degradation and provide a binding st that can be used for aft purification The protein q of imeest can sometimes be recovered in an active form by inloding a proteolytic cleavage site at an tpproprnte location in the fsion protein, During the ast 20 years, many diferent carer sequences have been used in hybrid proteins. Anyone ‘of them may work wel in a specific constructor in a particular bacterial strain, but none of them isa uni- versal panacea, However, newly developed expression systems that utilize F, cal protein thioredoxin as acar- rier appear to be a significant improvement over more traditional fusion partners, such as B-galactosidase. ‘The following are the two most problematic groups of proteins. ‘© Gytosolic proteins >100 residues are sometimes toxic, often unstable, and frequently form insoluble } inclusion bodies. }Information Panels 15.57 + Large cell-surface proteins and secretory proteins are translocated inefficiently across the inner mem- brane of & col, and only a small fraction of the molecules that reach the periplasmic space are able t0 fold into an active configuration. Perhaps this is not surprising in view of the fact that in mammalian cells, transport, folding, and biological activity of cell surface and large secretory proteins are often dependent on the coreect constellation of chaperone proteins, as wel as glycosylation and/or other forms ‘of posttranslational modification. In the absence of any informing principles that would help to find a general solution to the problems ‘of intracellular protein folding, the expression of every eDNA encoding “difficult” proteins in E.coli pre- sents a unigue set of problems, which must be solved empirically LACZ FUSIONS Gene fusions are created by joining together two pieces of DNA that were not previously connected. Often, the regulatory sequences of the gene of interest are placed upstream of sequences coding for a reporter mol- ecule whose level of expression can be easily measured, for example, galactosidase. This type of gene fusion allows rapid experimental analysis of the physiological conditions affecting the expression of the gene of interest; and it facilitates mutational analysis of the upstream regulatory region. Gene fusions are also used to create hybrid proteins in which the protein of intrest i attached to the amino terminus or the carboxy! ‘terminus of a carrier protein. Many vector systems have been developed for the expression of lacZ fusion genes. Some of these are listed in Table 15-6, LacZ fusion proteins have several advantages: ‘+ Their expression is placed under the domsinion of a well-characteized promoter whose activity can be increased by several onders of magnitude by simple manipulation of the bacterial culture. Transcription of LacZ is subject to both negative and positive regulation, Negative regulation is exerted by the Lac repressor, which binds to the operator (LacO) and prevents synthesis of lac mRNA. The lac repressors a tetrameric protein of 150 KD, is encoded by the lac! gene. Normally, there are about ten molecules of repressor per cel sufficient to smother, but not completely strangle, transcription of the Lac operon. In the absence of inducer, basal expression generates ~60 molecules of the monomer of B-galactosidase per cell when lacZ scarred asa chromosomal F gene, an higher levels when the gene is carried on a mul- ticopy plasmid, Because this level of basal expression may lead to problems if a LacZ fusion protein is ‘expressed that storie to E.coli, many LacZ fusion systems make use of a mutant lal gene (lat Maller- Fill etal. 1968) that synthesizes about tenfold more repressor than wid type. This overproduction of repressor suppresses the basal level of transcription of the Lac operon in the absence of inducer, Repression of the LacZ. operon is relieved by inducers, such as IPTG, which bind to the repressor and cause a conformational change that lowers the affinity of the repressor forthe lac operator. A fully induced culture of E.coli contains ~60,000 molecules of lacZ B-galactosidase per cel {In addition to an inducer such as IPTG, the efficient expression of lacZ requires CAMP and an ati: ‘ator protein, CAP. The cAMP-bound form of CAP binds with high affinity toa stein the lac promot- er (Majors 1975) and facilitates binding of RNA polymerase. Because CAP acts in a positive fashion, this type of regulation is called positive contol. Inthe absence of CAP, the levels of lic gene products are reduced up to 50-fold (Beckwith et al. 1972). The activity of CAP is controlled by the intracellular level ‘of cAMP, which sin turn, regulated by the concentration of glucose. Growth of cells on glucose reduces ‘the intracellular concentration of cAMP and inhibits induction ofthe lac operon, ‘©The fusion protein inherits aribosome-binding ste and initiation sequence whose efficiency is proven. lacZ does not have a particularly good Shine-Dalgarno sequence, and the sequences immediately sur rounding the start codon are not optimal for initiation of translation (Hui etal. 1984). Nevertheles.ini-15.58 Chapter 15 Expression of Cloned Genes in Escherichia coli tiation of protein synthesis occurs efficiently enough that it is not normally a limiting factor in expres sion of LacZ fusion proteins 1¢ The lacZ segment of the fusion protein serves as an identification tag that can be use for affinity purifi- cation on Sepharose columns containing the immobilized inhibitor of f-galactosidase, p-aminophenyl- B-D-thio-galactoside (also known as TPEG or APTG) (Steers et al. 1971; Germino et al. 1983; Ullmann 1984), 4 Fusion to acZ offers some protection agains the intracellular proteolysis that lays waste to many foreign proteins expressed in E, col, Produced on theie own, these proteins evidently fold into conformations that are susceptible to degradation by ATP-dependent proteases such as fon and clp (Gottesman 1989} This fate is particularly true of small polypeptides such as somatostatin (Itakura et al. 1977), insulin (Goeddel et al. 1979), and B-endorphin (Shine et al. 1980). When attached to lacZ or another caries, these proteins are stabilized tothe point where the fusion protein can comprise 20% of the total cell pro tein (for review, please sce Marston 1986) ‘© Finally, the high levels of expression that can be achieved with LacZ fusion proteins promote the forma- tion of insoluble aggregates or inclusion bodies, which can be isolated easily (Marston 1987). However, to obtain active fusion protein, the inclusion bodies must be dissolved in strongly denaturing buifers, and the fusion protein must then be purified and refolded. These stepsare not trivial, and conditions for cach of them must be developed empirically: For reasons that ate not understood, foreign proteins attached to the carboxy! terminus of lacZ are usually sequestered into inclusion bodies; amino-terminal fusion proteins are generally soluble and can be directly purified from cell lysates by affinity chromatog: raphy. Like other expression systems, LacZ fusions have some disadvantages. Fitstonly a small fraction of the amino acid cesidues of the fusion proteins may belong to a foreign protein, Second, to obtain biologically active material, the foreign protein usually must be cleaved, either enzymatically or chemically, from its LacZ carrier. Ths cleavage can be accomplished by building a unique cleavage site between f-galactosidase and the fusion protein, However if the fusion protein is insoluble, cleavage may be possible oly alter denat tration or in the presence of denaturant. This feature places restrictions on the types of enzymatic cleavage that can be used, Third, f-galactosidase is not a secreted protein and cannot be translocated across the ‘membrane of E.coli even if tis equipped with a signal sequence. Typically LacZ. fusion proteins that carry functional signal sequences become jammed in the membrane and are toxic tothe cell (Bassord ct al. 1979; Moreno etal. 1980). Despite these disadvantages, lacZ fusions were used successfully in the 1980s to express more than 50 proteins in E. cal. Nowadays, however, lacZ would probably not be the system of choice. Vectors containing [promoters for bacteriophage T7 RNA polymerase have tighter transcription control and can drive higher levels of expression (Studier etal, 1990), Polyhistcine tracts (Smith et al, 1988) may now be the fist choice for protein tags since they generally do not destroy protein function and allow affinity purification to be ar ried out under gentle conditions,TABLE 15-6 Systems for Expression of LacZ Fusion Proteins SSITE OF INSERTION PLASMID VECTOR OF FOREIGN DNA PROMOTER Information Panels 15.59 Comments ReFERENCES: PUR series } UK series DEX series pMRIO0 PAX series t UC series, PGEMZ series, Bluescript series, and many others ‘arbon terminus of lacZ of lacZ carboxy terminus of lacZ carboxyl terminus of lacz of fragment of Begalactosidase WV5lac vvSlac bacteriophage 4p, UVSlac (2 copies) ae lac Contains multiple doning ses inall three reading frames. Contains cloning sites in all three reading frames. Contains a cloning site in all three reading frames. ‘When forcign DNA is inserted into the polycloning sit, a fasion protein is sytesized that has the amino terminus of the bacteriophage Ac gene, a central segment of foreign Protein, and p-galactosidase at the carboxy terminus Atthe end ofthe lacZ gene, PAX vectors contain (1) hinge region of 187 bp encoding a fragment of collagen, (2) an endoprotein: ase Xa recognition site followed by (3) a multiple cloning ste and (4) the bacteriophage & transcriptional terminator. Contains multiple doning sits in all three eeading, frames; insertion of foreign DNA in any frame abolishes «e-complementation; inser tion in the corret reading frame generates a Lac. «fragment fusion protein Riither and Mille Hill (1983 Koonen etal. (1985) Stanley and Luzio (1984) Gray etal. (1982), Germino and Bastia (1984) ‘Messing (1983); Nor- sander ef al. (1983); Yanisch-Perron etal, (1985) Product literature from Stratagene and Promega ‘UVSlar iss double mutant of the lac promoter in which Go A wantin es adjacent oa T to A transversion inthe RNA polymeras-inding te Via ean “up” promoter that lls transcription of the lar operon in the absence af the CAF-AMP complex (Dikson et 1975),