rpplied
`icrohiology
`Biotechnology
`
`and
`
`A 20605
`
`~---------------------
`
`Volume 46
`
`Number 1
`
`August 1996
`
`Aho S, Arffman A, Korhola M: Saccharom yces cerevisiae
`muta nts selected for increased production of Tricho(cid:173)
`derllla reesei ce llulases 36
`Gellissen G, Piontek M, Dahlems U, Jenzelewski V, Gavagan
`JE, DiCosimo R, Anton DL, Janowicz ZA: R eco mbina nt
`Hansenula po/ym orpha as a bioca ta lyst: coex pression
`of the spin ach glyco late oxidase (GO) and the S. cerevi(cid:173)
`siae ca ta lase T (CTTZ) ge ne 46
`Vazquez-Cruz C, Ochoa-Simchez JC, Olmedo-Aivarez G:
`Pulse-fie ld ge l-e lectrophore tic analysis of th e amplifi(cid:173)
`cation a nd copy- numbe r sta bility of an integratio nal
`pl asm id in Bacillus subtilis 55
`Sugiyama M, Yuasa K, Bhuiyan MdZA, lwai Y, Masumi N,
`Ueda K: IS43lm ec-mediated integration of a bl eo mycin(cid:173)
`resistance ge ne int o the chromosome of a me thicillin(cid:173)
`resista nt S!{tphy lococcus aureus stra in iso lated in J apa n
`(S ho rt co ntribution ) 61
`
`(Continued on inside)
`
`Mini-Review
`
`Sawers G, Jarsch M: Alte rn a tive regulation principl es for
`the product io n of recombinant proteins in Escherichia
`coli 1
`
`Biotechnology
`
`Yang Z, Suzuki H, Sasaki S, Karube 1: Disposable senso r
`for bi oche mi ca l oxygen de mand 10
`
`Biochemical engineering
`
`Katoh T, Kikuchi N, Nagata K, Yoshida N: A mut a nt tryp(cid:173)
`sin-like e nzyme from Streptom yces .fradiae, crea ted by
`sit e-direc ted mutagenes is, improves affinity chro mat o(cid:173)
`graphy for protein trypsin inhibitors 15
`Garman J, Coolbear T, Smart J: Th e effect of cations on
`the hydrol ys is of lactose and th e transfe rase reactions
`catalysed by .B-ga lactosidase from six stra ins of lactic
`acid bacteri a 22
`
`Applied genetics and regulation
`
`Gouka RJ, Hessing JGM, Punt PJ, Starn H, Musters W, Van
`den Hondel CAMJJ: A n ex pressio n sys te m based o n the
`I ,4-.B(cid:173)
`promote r regio n o f th e Aspergillus m va1110ri
`e ndoxyla nase A ge ne 28
`
`Springer
`
`BEQ 1022
`Page 1
`
`

`
`Applied
`and - Microbiology
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`BEQ 1022
`Page 2
`
`

`
`Appl Microbia l Bio technol ( 1996) 46: 1- 9
`
`MINI-REVIEW
`
`f Sprin ge r- Ve r lag 1996
`
`G. Sawers · M. Jarsch
`Alternative regulation principles for the production
`of recombinant proteins in Escherichia coli
`
`Received: 17 Novembe r 1995/ Rece ivecl rev isio n: 9 February 1996/ Acceptecl: 4 March 1996
`
`Abstract Esta blished ex pression vec to rs ex ploiting
`reg ulated promoters such as the lac or toe promoters
`have eco nomi c a nd technical limitations when used for
`th e industrial produ ctio n of recombinant proteins.
`Co nsequently, a lterna ti ve expressio n systems are being
`developed that can be more rea dil y manipulated while
`m a intaining hi gh yields of protein. Several suitable
`ex pression vecto rs ha ve bee n described for use in Es(cid:173)
`cherichia coli that a re based on promote rs the activity
`of which is und er metabolic contro l. This a rticl e di s(cid:173)
`cusses the advantages a nd disadvantages of a cross(cid:173)
`secti o n of these expressio n sys tems, how they compare
`with esta blished sys tems a nd how th ey ca n be app lied
`to the industrial-scale production o f recombinant pro(cid:173)
`teins.
`
`Introduction
`
`A number of criteria mu st be considered ;vhen optimiz(cid:173)
`ing conditions fo r th e la rge-sca le ove rproducti o n of
`a recombinant protein. These cover the stabilit y of the
`mRNA (Gross 1989), the efficiency of mRNA transla(cid:173)
`tion (Gold and Stormo 1990) the accuracy of a mino
`acid incorporation (Santos and Tuite 1993), whether
`th e protein is correctly fold ed (Hackney 1994), the
`formation of inso luble protein aggregates (inclusion
`bodies) (Hackney 1994), the susceptibility of the prod(cid:173)
`uct to proteolys is (Go ttesman 1990; Nygren et al. 1994)
`or the requireme nt for pos t-translationa l modification
`
`G. Sawers
`N itrogen Fixa tio n Labora to ry, John Innes Centre.
`o rwic h R4 7U H, UK
`
`M. Jarsc h ( BI )
`Di v. The rape uti ka, F & E Bi o techn ologie,
`Boehringer Mannheim G mbH . Werk Pe nzbe rg.
`No nncnwa ld 2, D-82372 Pe nzbe rg, Germa ny. ~
`Fax: (49) 8856602 144
`
`such as proteolytic processing or phosphorylation, and
`whether the product must be ex ported to produce an
`active protein (Hackney 1994; Missiakis et a l. 1993). All
`of these criteria must be considered for each produ ct
`indi vidua ll y. The choice o f promote r used in a vector
`sys tem has a major bea rin g on ma ny, but not a ll, of
`these criteria. Hence, factors such as inclusion-body
`formation , the freq uency of mistransla lion eve nts or th e
`correct folding of a protein can be influenced acco rding
`to how th e activity of th e promote r is regulated. Fur(cid:173)
`th ermore, use of a st ron g promoter th a t produces large
`amounts o f mRNA substra te can compensa te in part
`for mRNA in sta bility, poor tran slation efficiency or a n
`unsta ble product. Prudent use of a promoter, the refore,
`ca n have a fundamental impact on the quality and yie ld
`of a recom binant protein while co ncomita ntl y minimi z(cid:173)
`ing both developme nt and production costs. In this
`review we sha ll describe several metabolicall y regulated
`promoters that have been successfull y used in exp res(cid:173)
`sio n vec tors for the la rge-sca le production of reco m(cid:173)
`binant prote ins in E. coli. The activity of th ese pro(cid:173)
`moters res pond s to flu ctu a tions in metabolism brou g ht
`about by manipulating cultivation parameters such as
`the concentration of phosp hate or oxygen . Co nse(cid:173)
`qu entl y, exp ressio n vectors th a t include metabo licall y
`regul a ted promoters provide cost-effective alternatives
`to es tablished ex pression system s ba sed on ,
`for
`exa mpl e, the lac o r tac promoters.
`
`Categories of recombinant protein
`
`In essence, there are three major ca tegories of recom (cid:173)
`binant proteins currently produced o n an indust ri a l
`sca le, and th ese are listed in Table 1. T he first ca tegory
`includes
`tec hnical enzymes and proteins for food
`processing where it is not critical that the product is
`absolutely pure but hi gh yield is an important factor
`(Richter 1986). Therefore, since minimal down strea m
`processing is advanta geo us it is preferable that th e
`
`BEQ 1022
`Page 3
`
`

`
`2
`
`Table I Principle categories of recombinant protein
`
`Catego ry
`
`Qualitative and
`Quantit ati ve
`prerequisites
`
`High yield
`Low cost
`Purity is not a
`major concern
`
`2
`
`3
`
`High purity
`Low deve lopment
`error! and cost
`Yield less critical
`tha n for ca tego ry I
`
`Very high quality
`Consistency in
`production
`Process must be
`compatible with
`Food a nd Drug
`Administration
`guidelines
`
`Applica tion
`
`Technica l enzymes, e.g.
`proteases and lipases for
`washing powders
`Proteins for food
`process ing
`or supplementation , e.g.
`glucose ox idase
`Bioca talysts, e.g. glucose
`ox idase
`
`Enzymes and proteins for in
`vivo diagnostics, e.g.
`cholesterol oxidase, glucose
`dehydrogenase or
`penicillin-G acylase
`
`Human therapeutics, e.g.
`recom bi nan t tissue
`plasminogen activator,
`insulin
`
`protein is produced in high amounts, of the order of
`20% - 40% of the total cellul ar protein. For thi pur(cid:173)
`pose it is necessary to have an expression vector with
`a strong promoter. In the other two categories there is
`a st ronger emphasis on the quality of the product,
`quantity bein g a seco ndary consideration (Kopetzk i et
`al. 1994). This is particularly relevant for production of
`therapeutics where a consistently high-quality product
`is a prerequisite. Again, this can be affected by the
`promoter driving expression particularl y, how strong
`the promoter is and how its activity is controlled. The
`protein-synthetic capacity of a cell limits the amount,
`and to a certain extent the quality, of a recombinant
`
`protein that can be sy nthesized. Therefore, although
`under ideal circumstances it would be desirable to have
`a host ce ll that grows rapidl y and attains very high cell
`densities before synthesis of the recombinant protein is
`switched on , this is not always practica ble beca use the
`host cell must deliver sufficient ATP and metabolic
`intermediates to synthesize large amounts of a recom(cid:173)
`binant protein. Moreover, at very high cell densities
`nut rients become limiting, which can lead to misincor(cid:173)
`poration of amino acids (Santos and Tuite 1993) or
`premature termination of polypeptide chain elongation
`(Balbas and Bolivar 1990). Hence, it is necessary to be
`ab le to control promoter activity easi ly and efficiently
`so that synthesis of the recombinant protein is optimal.
`
`Relevant features of promoters in expression vectors
`
`T here are both essentia l and desirable features of a pro(cid:173)
`moter for use in an expression vector (Table 2). It is
`essential when the promoter is activated that formati on
`of the open transcription complex is efficient and that
`promoter clearance is rapid. This ensures that large
`amounts of mRNA are synthesized. The promoter
`should have suA1cient strengt h to ensure that, und er
`optima l conditions, the ultimate product can attain
`levels greater than 10% of the total cellular protein,
`and the promoter should be regulated. The form this
`regulation takes, the "tightness" (i.e. the extent to which
`promoter activity can be preve nted) of that regulation
`and the extent to which promoter activity can be in(cid:173)
`duced (the induction ratio) are important consider(cid:173)
`ations.
`Transcription initiation from a promoter can be
`regulated either positively or negatively (Fig. 1). Posi(cid:173)
`ti ve regulation means that a specific activator protein
`must be present either to permit RNA polymerase to
`initiate transcription or to increase the frequency of
`transcription initiation (Gralla J 990). The activity of
`
`Table 2 Feat ures of a 1 romoter
`desirable in an express ion vector
`
`Salient fea tures of the promoter Alternatives. va riables or req uirement s
`
`Location and stabilit y
`
`Strengt h
`
`Regulation
`
`Activat ion
`
`Mode of controlling act ivation
`
`I. High-copy-number plasmid
`2. Transcription should not interfere with plasmid
`replication
`I. Slow initia tion and rapid elonga ti on. e.g. l11cU 115
`promoter
`2. Fast initiation and very rapid elonga tion, e.g. phage T 7
`promoters
`I. o promoter activity until product is desired
`2. Low-level expression from promoter during growth
`followed by co ntrolled activation
`I. Remova l of a represso r
`2. Act iva tion of a pos iti ve control fac tor (acti va tor)
`I. Tempera ture-shift inacti va tion of a represso r molecule
`2. Chemica l induce r added to the culture medium
`3. N utrient depri va tion all owing derepression or activation of the
`promoter
`
`BEQ 1022
`Page 4
`
`

`
`@~~
`
`A Positive regulation
`
`t
`- ~
`
`0
`
`p
`
`J
`
`'Metabolic signal'
`
`0
`
`(F
`~_,._
`
`p
`
`B Negative regulation
`
`(RpoD
`
`Control by induction
`
`Addition of inducer
`
`...
`
`®
`~ p
`
`C Negative regulation
`
`R+C
`
`Removal of
`
`Control by derepression -----~_..,..__
`
`Co-repressor
`
`I
`~
`
`R
`~
`
`p
`
`p
`
`F ig. lA-C Schematic rep resenta ti o n of common modes o f promo ter
`regula ti o n. A Positive regu lat io n. RNA polymerase (RPol) ca n int er(cid:173)
`act with the promoter (P) but cannot form an "open" complex (melt
`dupl ex DNA a nd initia te tra nscripti o n) because the activator A; is in
`th e inacti ve form. Upo n respo ndin g to a metabo lic signa l, e.g.
`a change in oxygen, nitrogen or p hos phorus concentration, A;
`und ergoes a con fo rmationa l cha nge to its active confo rm a tion, A,.
`
`A, can then bind to a specific operato r sequence (0; •l. which is
`
`loca ted upstream of th e promoter on the DNA. By maki ng contact
`with RNA po lymerase facilitated through DNA "looping" the ac(cid:173)
`tivator can pro mote open complex fo rmation a nd transcri ption
`initiation. Two modes o f nega ti ve regula ti o n a re depicted. B In
`control by inducti o n, the interac tio n of RNA polyme rase with the
`promoter is prevented by binding of the rep resso r protein (R) to its
`o perator site, which in this case overlaps th e promoter. The re pres(cid:173)
`so r has a much higher affi nit y for its operator than RNA polymerase
`has for th e promote r. The repressor-inducer (I) complex has a dras ti(cid:173)
`cally reduced affinity fo r the o pera to r allowing access of RNA
`po lymerase to th e promoter. C In the case of contro l by derepres(cid:173)
`sion, the scena ri o is simil ar to that depicted in B except th a t the
`repressor must interact with a sma ll mo lecule (C = co- represso r) to
`enha nce its affi nit y for its operator. Me ta bo lism of the co-repressor
`inacti va tes th e rep ressor a nd libera tes the promo ter.
`
`the activator protein is usuall y controlled in response
`either to a cha nge in the meta bolic status of the cell or
`to the add ition of a specific inducer molecule. Negative
`regulation means that transcription initia tion from
`a promoter is prevented by a repressor molecule (usu(cid:173)
`a ll y a protein). The ,activity of a nega tively regulated
`promoter, in turn, can be co ntrolled in two ways. In
`one instance the promoter is controlled by induction,
`e.g. the lac promoter (Makoff and Oxer 1991), where
`the addition of the inducer lactose prevents repressor
`binding, thus allowing transcription to proceed. In con(cid:173)
`trast, the trp promoter (Squires et al. 1975), for exa mple
`is controlled by repression. Here tryptophan is a co(cid:173)
`repressor and, when it becomes depleted , the repressor
`can no longer bind to the promoter and transcription
`can occur. How tightly a promoter is regulated depends
`principally to what extent transcription still occurs in
`
`the presence of a represso r or in the absence of a n
`activator. Hence, if the recombinant protein is toxic to
`the host it is necessary that the promo te r is ve ry ti ghtl y
`regulated (Wi.ilfing and Pli.ickthun 1993). In the case of
`plasmid-based promoters controlled by repression this
`may require that extra copies of the ge ne encoding the
`repressor are also supplied on a plasmid to prevent
`repressor titration (Stark 1987).
`There are several other features of a promoter that
`may be desirable but thi s will depend very much on the
`quality of the product, the quantity of the product that
`is required and whether the product is to xic to the host
`cell . These features a re listed in Ta ble 2.
`
`Expression systems
`
`Established expression systems
`
`Strong, regulated promoters commonly employed in
`both research la boratories and industry to drive het(cid:173)
`erologous ge ne expression include the promoters from
`the lac operon and the tryptopha n (trp) biosynthetic
`operon, as well as phage promoters such as the ), Jh
`promoter and
`the c/J10 promoter from phage T7
`(Table 3). All of them have been used to produce large
`numbers of recombinant proteins that attain levels of at
`least 5%- 10% of the TCP. The trp and lac promoters
`are both nega tivel y regulated and have been used suc(cid:173)
`cessfull y to overproduce a vast number of recombinant
`proteins (Balbas and Bolivar 1990; Tacon et al. 1980;
`Yansura and Henner 1990).
`Hybrid promoters, combining different portions of
`the lac and trp promoters have been constructed and
`used to design improved ex pression vectors compared
`with those based a round the natural promoters. Exam (cid:173)
`ples include the tac, trc and tic promoters (DeBoer et al.
`1983; Brosius et al. 1985) which combine the -35 RNA
`
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`
`

`
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`_o--
`~ -
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`<
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`:J
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`on e
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`
`polymerase recognition region from the trp promoter
`with a ca no nica l - 10 RNA polymerase recognition
`sequence and the Lacl operator from the lac promoter.
`The three promoters differ in the spacing between the
`- 35 and
`- 10 seq uences which a ffects promoter
`strength. The we promoter is the most efficient of the
`three, being fi ve times stron ger than /a cUV5 and it still
`retai ns the regul ation by Lacl (D eBoe r et a!. 1983;
`Brosius et a!. 1985).
`Although these expression systems are used in indus(cid:173)
`try there are several shortcomings that detract from
`their positi ve attributes. As mentioned above, temper(cid:173)
`ature shifts ma y cause locali zed overheating within th e
`fermenter and they are difficult to control. Such shifts
`have the added disad va ntage that correct protein
`foldin g is often impaired and they can exacerbate the
`risk of proteolysis (Hockney 1994). Inclusion-body
`formation also can be increased by te mperature shifts;
`however, this is not always undesirable and depends
`very much on the product. Owing to the increased
`number of operator sites in plasmid-based expression
`systems it is often necessary to increase the number of
`represso r molecules. This can be ach ieved by introdu(cid:173)
`cing the ge ne encod in g the repressor onto th e ex pres(cid:173)
`sion plasmid itself or by using a second plasm id. It is
`possible that either of these so lutions may reduce host
`cell growth rates a nd yields or it might decrease the
`genetic stability of the expression system (Balbas and
`Bolivar 1990). Addition of inducing agents, such as
`indoleac rylic acid or iso propylthiogalactoside is ex pen(cid:173)
`sive and they must be distributed quickly and evenly
`throughout the fe rm enter. Also, care mu st be taken to
`ensure th a t they are completely removed from the
`product at the processing sta ge, since their concentra(cid:173)
`tion ca n be relatively hi gh , they are not metabolized
`and it is possible that they co uld co-purify with recom(cid:173)
`binant proteins.
`
`Alternative expression systems
`
`Alternative expression vectors that include promoters
`the control of which may be more tractable to the
`cost-effective production of recombinant proteins on
`an industrial sca le have been developed in recent years.
`The promoter shou ld fulfil the criteria listed in Table 2.
`Ideally, activation should occur after minor adjust(cid:173)
`ments have been made to th e fermentation conditions,
`for example by altering the flow of oxygen to the
`cu lture or by taking advantage of nutrient limitations,
`such as carbon, phosphorus or nitroge n so urce de(cid:173)
`pletion , which occur during the fermentation process.
`The next sections describe severa l promoters exhibiting
`metabolic control that ha ve been examined for their
`ca pacity to provide possible alternatives to established
`systems. The characteristics of these promoters are
`summarized in Table 4 and their mode of regulation is
`shown in F ig. 2. In some cases the efficacy of the
`
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`
`

`
`Ta ble 4 Expression systems unde r metabolic control
`
`Promoter
`
`In duction principle
`
`1nducti on
`rat io
`
`Yield( % ) of References
`tota l cellul a r
`protein
`
`5
`
`phoA alka line phosphatase
`II(Jfl Sll-glycerol-3-phos ph ate
`trans1 ort operon
`ara B ara bin ose operon
`
`Ph os phate de pri vati on
`Phos phate depri vation
`
`> 1000-fold
`~ 100-fold
`
`20- 60
`50
`
`G lucose depleti on and
`arabin ose add ition
`Gl ucose add ition
`
`111gl meth yl ga lactoside
`transport operon
`Phb Vitreoscil/a haemoglobin gene M icroaerobiosis
`Anaerobiosis
`nirB nitri te reductase operon
`
`1200-fold
`
`15
`
`~ 100-fo ld
`
`>50
`
`30- fold
`100- fold
`
`15
`> 30
`
`Wanner 1993: Carter et al. 1992
`vVa nner 1993; Su et al. 1990. 1991;
`Kasa hara et al. 199 1; Jarsch un published
`Ca gnon et al. 199 1; Lobell and Schleif 199 1
`
`Sch umacher et al. 1988; iVliiller 1989; Deat h
`and Ferenci 1994; Jarsch, un published
`Khos la and Ba iley 1988; Dikshit et al. 1990
`Cha rles et al. 1992; C hatfield et al. 1992:
`Schroeck h et a I. 1992
`Sawers 1993: O xer et al. 199 1
`
`nfl pyruva te fo rmate-lyase operon A m1erobiosis
`
`25- to 30-fold
`
`> 40
`
`prom o ter for use in a n ex pressio n vect o r has bee n
`tested fo r o nl y o ne o r t;vo produ cts. H oweve r, this
`suffices to give a n ove ra ll impressio n o f th e qu a lities
`a nd limita ti o ns of each a nd th eir p o tenti a l fo r fu ture
`develo pment.
`
`Promoters
`
`N utri ent-reg ul a ted prom o ters
`
`T he gene encod ing alka line ph osph a tase (phoA) is ex(cid:173)
`pressed in E. coli at very hi g h levels when cells a re
`sta rved of ino rga ni c phosp hate (Wa nn er 1993). E xp res(cid:173)
`sio n ca n be induced mo re th a n 1000-fold with Ph oA
`a tt ainiog levels o f up to 6% of th e tota l cellul a r protein
`from a chromosom a l cop y of th e phoA ge ne. The phoA
`pro moter is regul a ted bot h positi ve ly a nd nega ti ve ly by
`the PhoB pro tein in res po nse to a lte ra ti o ns in the
`in o rga nic p hos ph a te co ncentrat io n (T a ble 4, F ig. 2)
`(W a nner e t a l. 1988). Pho B is th e regulato r co mpo nent
`o f a two-co mpo nent sig na l-tra nsdu cti o n cascade and
`w hen the in o rga ni c ph os pha te co ncen tra ti o n [Pi] is
`a bove 5 mM , Pho B bind s to the prom o ter a nd p re(cid:173)
`ve nts tra nsc rip tio n. If [PJ dro ps below 1 m M Pho B
`becomes phosp ho ryla ted by th e Pho R histidin e kin ase,
`whic h co nve rts Ph o B protein into a transc ription a l
`acti va tor. Specific deta ils of Ph o regul o n co ntro l have
`bee n reviewed recently (Wa nn er 1993).
`T he plw A pro mote r has bee n used to co nstru ct phos(cid:173)
`ph ate-regul a ted ex pressio n vecto rs th a t have been suc(cid:173)
`cessfull y empl oyed to produ ce reco mbin a nt protein s.
`Two exa m pies incl ude th e prod uctio n o f hum a n epider(cid:173)
`ma l g row th facto r (Oka e t a l. 1985) a nd hum a ni zed
`Fa b' fragments (Ca rter et a l. 1992). In the la tter st ud y
`the co nce ntra ti o n o f Fa b' secre ted into th e culture
`medium atta ined level s of 1- 2 g l- 1
`.
`T he pro mo te r fr om the ugpB AECQ o pero n, encod(cid:173)
`ing a n sn-glyce ro l-3-phos ph a te tra nsport sys tem, is a lso
`
`acti va ted by Ph o B ph osph a te in respo nse to phosph a te
`sta rva ti o n (Wa nn er 1993; Su et a l. 1990). Max im a l
`pro mo ter acti va ti o n
`is a lso depend ent o n cAM P
`rece pto r protein (C RP) (Kasah a ra e t a l. 199 1; Su et a l.
`1991). Redu cti o n in bo th the phospha te a nd glucose
`co nce ntra ti o n is th e refo re required to acti vate ugp
`pro m o te r tra nscripti o n. A pBR322-based ex pressio n
`vec to r has bee n developed (S u et a l. 1990) whi ch is
`a pplicabl e to la rge-sca le ferm entatio ns (Ta ble 4). T he
`ugp prom o ter is ve ry stro ng a nd ex hibits bo th equi va