79

Recombinant DNA technology

T h e scope and br e a dt h o f b i o c h e mi -
cal e n g i n e e r i n g ha ve c ha nge d dr a -
mat i cal l y o v e r t he last t en years. Bi o -
chemi cal e ngi ne e r i ng has mo v e d far
b e y o n d its t r adi t i onal d o ma i n - t he
appl i cat i on o f c he mi c a l pr ocessi ng t o
i ndust ri al f e r me nt a t i ons - t o t he
p o i n t wh e r e it n o w plays a k e y r ol e
b o t h i n t he di s cover y and c o mme r -
cial d e v e l o p me n t o f n e w bi ol ogi cal
pr oduct s . A r e c e nt me e t i ng*, o r g a n -
i zed i mp e c c a b l y b y J u a n As enj o
( Uni ver s i t y o f Re a d i n g , Re a d i n g ,
UK and Un i v e r s i t y o f Chi l e,
Sant i ago, Chi l e) , c o v e r e d a b r o a d
s p e c t r u m o f e me r g i n g t e c hnol ogi e s
at t he i nt erface b e t we e n bi ochemi cal
e ngi ne e r i ng and mo l e c u l a r bi ol ogy.
Inevi t abl y, mos t o f t he 30 oral pr es en-
t at i ons a nd 50 post er s t i l t ed mo r e
heavi l y in one or t he o t h e r di r ect i on.
Never t hel es s , it was appar ent t hat t he
t wo fields are al r eady c o n v e r g i n g
r api dl y and t hat mo s t o f t he di f f er -
ences i n t he cul t ur es o f t he 6ngi n-
e e r i ng and bi ol ogi cal c o mmu n i t i e s
have al r eady b e e n o v e r c o me . I n this
r e por t , I ha ve sel ect ed s o me o f t he
pr es ent at i ons t hat , i n my opi ni on,
r ef l ect t he di ver si t y o f r esear ch
appr oaches unde r t a ke n. Th e r e we r e
ma n y o t h e r c ont r i but i ons o f equal l y
hi gh qual i t y t hat space does not al l ow
me t o cover .
T h e me e t i n g b e g a n wi t h t wo
excel l ent pl e na r y l ect ur es de l i ve r e d
by A. Pl f i c kt hun ( Uni ve r s i t y o f
Zi i r i ch, Zi i r i ch, Swi t zer l and) a nd
J. E. Bai l ey ( ETH, Z/ i r i ch, Swi t zer -
land). Andr eas Pl f i c kt hun di scussed
t he e ngi ne e r i ng o f i mmu n o l o g i c a l
r ecept or s a nd t hei r expr es s i on i n
E. coil Hi s g r o u p has successful l y
used pr ot e i n engi neer i ng, mol e c ul a r
b i o l o g y and cell p h y s i o l o g y
appr oaches t o i ncr ease t he expr essi on
o f s i ngl e- chai n Fv ant i bodi es. N o t
c o n t e n t wi t h t he i r successes wi t h
i nmmnogl obul i ns , Pl f i ckt hun' s gr oup
r ecent l y d e mo n s t r a t e d t he bact er i al
expr essi on o f si gni fi cant a mo u n t s o f
cor r ect l y f ol ded T- c e l l r e c e p t o r f r ag-
ment s , a n o t h e r t ype o f i m m u n o -
l ogi cal r e c e pt or mol e c ul e . Pr o d u c i n g
f unct i onal s i ngl e- chai n T- c e l l r e c e p -
t ors p r o v e d t o be chal l engi ng a nd
*The meeting 'Recombinant 1)NA Biotech-
nolow: The Integration of Biological and
Engineering Sciences' was held in Deauville,
France, 16-21 October 1994.
was onl y possi bl e b y ma ni pul a t i ng
t he cel l ' s stress r es pons e and t he
abi l i t y t o f o r m disulfide bonds 1.
T h e ma n i p u l a t i o n o f cel l ul ar
me t a bol i s m was t he cent r al t h e me i n
J a y Bai l ey' s pl e na r y l ect ur e. Bai l ey
s u mma r i z e d t he several r esear ch
pr oj ect s t hat are b e i n g act i vel y p u r -
s ued b y his lab. A mo n g t he mo s t
fasci nat i ng is t he e ngi ne e r i ng o f t he
e uka r yot i c cell cycl e t o c ont r ol cell
pr ol i f er at i on u p o n de ma nd. Hi s
g r o u p s h o we d t hat over expr es s i on o f
cycl i n E can i nduc e t he pr ol i f er at i on
o f C H O cells in s e r u m- and p r o t e i n -
f r ee medi a. Ci r c u mv e n t i n g t he
r e q u i r e me n t f or p r o t e i n g r o wt h
f act or s affords gr eat cost r e duc t i ons
and s i mpl i f i cat i on o f tissue cul t ur e.
On g o i n g st udi es are n o w c o n c e n -
t r at i ng o n e ngi ne e r i ng t he cont r ol l ed
pr ol i f e r a t i on o f t er mi nal l y di f f e r e n-
t i at ed h u ma n cells, t o b e us ed f or
t he t r e a t me n t o f a var i et y o f disease
states.
T h e t h e me s o f p r o t e i n p r o d u c t i o n
in h e t e r o l o g o u s host s and o f
me t a bol i c e ngi ne e r i ng 2 we r e e c h o e d
i n wel l o v e r ha l f o f t he pr esent at i ons.
T h e d e v e l o p me n t o f n e w E. coli
p r o mo t e r s sui t abl e f or l ar ge- scal e
cul t ur es wi t h hi gh cell dens i t y was
r e p o r t e d b y L. Tsai ( AMGE N,
T h o u s a n d Oa ks , CA, USA) . An
o p t i ma l expr es s i on syst em f or
Fa b - g e l o n i n i mmu n o t o x i n s has b e e n
c ons t r uc t e d b y M. Be t t e r ( Xo ma
Co r p o r a t i o n , Sant a Mo n i c a , CA,
USA) . Th e s e f us i on pr ot ei ns c oul d
be p r o d u c e d i n sol ubl e f o r m at a s ub-
stantial l evel and exhi bi t ed hi gh c y t o -
t oxi c act i vi t y in vitro. T h e expr essi on
o f f usi on pr ot ei ns i n E. coli was also
r e v i e we d b y I. Mar czi novi t s ( Al ber t
Sz e n t - Gy 6 r g y i Me di c a l Uni ve r s i t y,
Szeged, Hunga r y) .
Co r r e c t f ol di ng o f r e c o mb i n a n t
pr ot ei ns is an i mp o r t a n t p r o b l e m i n
p r o t e i n expr essi on, par t i cul ar l y i n
bact er i a and yeast . F. Ba n e y x ( Un i -
ver si t y o f Wa s hi ngt on, Seattle, WA,
USA) pr e s e nt e d e vi de nc e t hat e x -
pr essi on o f Dn a K7 5 6 , a wel l char ac-
t er i zed mu t a n t o f t he bact eri al hsp70
c h a p e r o n e , can r esul t i n i ncr eas ed
sol ubi l i t y o f p r o t e i n fusi ons. G.
Ge o r g i o u ( Uni ve r s i t y o f Texas ,
Aust i n, T X, USA) discussed t he f or -
ma t i o n o f disulfide b o n d s i n pr ot ei ns
s ecr et ed i n t he bact er i al per i pl asmi c
space. I t t ur ns o u t t hat , unl i ke
e uka r yot i c cells, E. coli is def i ci ent i n
e nz yma t i c act i vi t i es capabl e o f cat a-
l yzi ng t he r e a r r a nge me nt o f disulfide
bonds , a pr ocess necessar y f or t he
c or r e c t f ol di ng o f ma n y
he t e r ol ogous pr ot ei ns . I n S. cerevisiae
t he f o r ma t i o n and r e a r r a n g e me n t
o f di sul fi de b o n d s is cat al yzed b y
p r o t e i n di sul fi de i somer ase. D. K.
Wi t t r u p ( Uni ve r s i t y o f Il l i noi s,
Ur b a n a , IL, USA) s h o we d t hat
c o- e xpr e s s i on o f p r o t e i n di sul fi de
i s omer as e results i n a si gni fi cant
i ncr ease i n t he expr es s i on o f s ome ,
b u t n o t all, h e t e r o l o g o u s pr ot ei ns 3.
O n t he o t h e r hand, ove r e xpr e s s i on
o f t he yeast Bi P c h a p e r o n e (t he yeast
hsp70 anal og) di d not ha ve an ef f ect
o n s ecr et i on l evel .
I nt er es t i n Bacillus f or p r o t e i n
expr essi on is pi c ki ng up agai n, f uel ed
b y t he G R A S (general l y r egar ded as
safe) status a nd t he ease o f p r o t e i n
e x c r e t i o n f r o m this p r o k a r y o t e . A
series o f stable pl asmi d vect or s f or t he
expr essi on o f e xc r e t e d pr ot ei ns f r o m
B. subtilis has b e e n d e v e l o p e d at
Du P o n t b y V. Nagar aj an ( Du Po n t ,
Wi l mi n g t o n , DE, USA) . Ext r acel l u-
lar r e c o mb i n a n t p r o t e i n l evel s wel l
o v e r 1 gl t ha ve b e e n de mons t r a t e d.
I n a s o me wh a t di f f er ent a ppr oa c h,
S. Ebi su ( Hi get a Shoyu Co. , Chos hi ,
Japan) and c o - wo r k e r s used B. brevis
f or t he l ar ge- scal e p r o d u c t i o n o f
h u ma n e pi de r ma l g r o wt h f act or
( hEGF) . Ev e n by Bacillus standards,
B. brevis is a ve r y pr of i ci ent e xc r e t e r
o f pr ot ei ns. Ebi s u' s g r o u p a c hi e ve d a
r e ma r k a b l e l evel o f 4 gl < o f e xt r a -
cel l ul ar h EGF. T h e y t h e n s h o we d
t hat t he r e c o mb i n a n t h EGF , wh e n
i nj ect ed in sheep, results i n c o mp l e t e
hai r loss and t hus affords a s i mpl e
t e c hni que f or wo o l sheari ng.
Pr ot ei ns r e qui r i ng c o mp l e x p o s t -
t ransl at i onal modi f i cat i ons c a nnot be
p r o d u c e d i n pr oka r yot e s or l o we r
eukar yot es . I n r e c e nt years, gr eat
pr ogr ess has b e e n ma d e i n t he
expr essi on o f he t e r ol ogous pr ot ei ns
i n cell lines de r i ve d f r o m hi ghe r
eukar yot es . Pr obl e ms wi t h hi gh va r i -
abi l i t y a nd l o w expr es s i on l evel s i n
t r ans f ect omas appear s t o ha ve b e e n
sol ved b y F. Wu r m and c o - wo r k e r s
( Ge n e n t e c h I nc. , Sout h San
Fr anci sco, CA, USA) . T h e y us ed
r et r ovi r al s equences pr e s e nt i n t he
g e n o me o f C H O cells as sites f or
t he c h r o mo s o ma l i nt egr at i on, via
h o mo l o g o u s r e c ombi na t i on, o f t r a n s -
f ect ed DNA. Wi t h this me t h o d , hi gh
l evel s o f r e c o mb i n a n t p r o t e i n e x-
pr essi on we r e obt a i ne d b o t h i n l a b-
o r a t o r y and l ar ge- scal e bi or eact or s .
W. H. Vi l l ander (Vi rgi ni a Po l y t e c h -
ni c I nst i t ut e, Bl acksbur g, VA, USA)
Meeting
report
1995, Elsevier Science Ltd 0167 - 7799/95/$9.50 TIBTECH MARCH 1995 (VOL 13)
80
f O r b l l f l
discussed the pr oduct i on o f Prot ei n
C in different transgenic animal
models. Prot ei n C is a gl ycoprot ei n
wi t h 12 disulfide bonds, one
~- hydr oxyl at ed aspartate and ni ne
glutamic acids that are y- c a r boxy-
lated in a vi t ami n K- dependent reac-
t i on 4. Villander and his colleagues
have showed that the pr oduct i on o f
Prot ei n C in the milk o f transgenic
pigs is the most promi si ng rout e for
commerci al purposes. However ,
there are several issues that still
need t o be addressed, t he most
i mport ant o f whi ch appears t o be
het erogenei t y o f post-translational
modifications.
Met abol i c engi neeri ng is defined
as ' t he i mpr ovement o f cellular ac-
tivities by mani pul at i on o f enzy-
matic, transport and regulatory f unc-
tions o f the cell '2. Clearly, metabolic
engi neeri ng represents t he next
frontier o f r ecombi nant DNA t ech-
nol ogy, a poi nt that was illustrated
persuasively by a number o f pres-
entations. W- S. Hu and D. H.
Sherman (both from the Uni versi t y
o f Mi nnesot a, Minneapolis, MN,
USA) discussed the engi neeri ng o f
antibiotic pr oduct i on in Streptomyces.
By per f or mi ng sensitivity analysis
on t he experimentally det er mi ned
parameters of a mathematical model o f
]3-1actam biosynthesis in S. clavuligems,
t hey wer e able t o det ermi ne the
rat e-l i mi t i ng enzymat i c step. Sub-
sequently, t hey showed that the
amplification o f t he gene encodi ng
the rate-limiting enzyme results in
increased biosynthesis o f cephal o-
sporin. A very exci t i ng approach
for the synthesis of novel polyketides
was out l i ned by D. H. Sherman:
new pol yket i de antibiotics were
pr oduced in St rept omycet es coelicolor
by replacing different subunits o f
polyketide synthetase wi t h homol ogs
f r om ot her species.
Impressive progress has been made
in engi neeri ng t he pri mary met ab-
olism o f the cell. E. T. Papoutsakis
(Nort hwest ern University, Evanston,
IL, USA) and co- wor ker s have
devel oped cl oni ng vectors and trans-
f oml at i on procedures and cl oned
several o f t he genes i nvol ved in
the pr oduct i on o f solvents and car-
boxylic acids by Clostridium acetobutyl-
icum. The genes encodi ng enzymes
i nvol ved in acet one or but anol for-
mat i on wer e assembled i nt o syn-
thetic operons whi ch were expressed
in various strains of clostridia t o alter
solvent format i on. B. Hahn-
H~igerdal (Lund University, Lund,
Sweden) described some o f t he
complexities that can be encount ered
in engi neeri ng cells t o utilize readily
available carbon sources. I n her
group' s wor k wi t h Saccharomyces cere-
visiae expressing het erol ogous xylose
reductase and xylitol dehydrogenase,
t hey f ound that xylose is efficiently
conver t ed t o xTlitol but the next
step i n its assimilation depends on
r edox levels wi t hi n the cell. G.
St ephanopoul os (MIT, Cambri dge,
MA, USA) discussed t wo examples
o f metabolic engi neeri ng for the syn-
thesis o f ami no acids. First, he
showed how t he carbon flux in
corynebacteria can be diverted away
f r om lysine biosynthesis towards the
pr oduct i on o f t hr eoni ne or iso-
leucine. Second, he present ed an
approach for identifying branch
points in cellular metabolism by em-
pl oyi ng met abol i c cont rol analysis
(MCA) wi t h successive rounds o f
experimentation. In parallel studies,
H. Sahm (Forschungzent mmJfi l i ch,
Jiilich, Germany) showed that ampli-
fication o f homoser i ne dehydr o-
genase and homoser i ne kinase in
corynebact eri a diverts the car bon
flux t o t he format i on o f t hr eoni ne
rather t han lysine. An impressive
study o f t he pr oduct i on o f pol y-
hydroxybut yrat e in E. coli was pr e-
sented by S-Y. Lee (KAIST, Taej on,
Korea). His gr oup achieved the pr o-
duct i on ofintracellular pol yhydr oxy-
but yrat e at mor e t han 85% o f the
total cell weight. The pr oduct i on o f
such hi gh levels o f bi opol ymers
resulted in cell filamentation s but this
coul d be ci r cumvent ed by over -
expressing a prot ei n (FstZ) i nvol ved
in sept um formation.
Several talks focused on processing
issues and the downst ream process-
i ng o f product s f r om genetically
engineered cells. Ne w strategies for
increasing the yield o f refolded pr o-
teins and prevent i ng aggregation
were present ed by E. Samuelson
( Royal Institute o f Technol ogy,
St ockhol m, Sweden) and J. B.
Chaudhur i (University o f Bath,
Bath, UK). The Royal Institute o f
Technol ogy gr oup observed that
fusion o f a prot ei n A fragment pre-
vent ed t he aggregation o f insulin-
like gr owt h factor-1 (IGF-1) upon
refolding. J. B. Chaudhur i discussed
the effect o f several parameters, such
as prot ei n puri t y and t he choi ce o f
denaturant, on refolding yields. He
also showed that extraction in
reverse micelles is a promi si ng
approach for efficient prot ei n refold-
ing. F. Matthiesen ( Novo Nordi sk
A/ S, Denmark) out l i ned different
strategies for prevent i ng reversible or
irreversible conformat i onal changes
duri ng the fol di ng o f r ecombi nant
proteins such as glucagon, IGF-1 and
human glutamic acid decarboxylase.
E. W. Lesser (University of P, eading,
Readi ng, UK) and the conference
chairman, J. A. Asenjo, are devel op-
i ng a database o f key physi co-
chemi cal properties o f t he most
abundant proteins in frequently used
hosts such as E. coli and C HO cells.
This database is used t o build an
expert system for selecting optimal
purification prot ocol s that allow the
efficient separation o f desired pr o-
teins from host component s whi ch is
certain t o pr ove very valuable, not
onl y for laboratory research but also
for teaching purposes.
The issue o f integration o f engi n-
eering and bi ol ogy research was the
subject o f a r ound- t abl e discussion
coordi nat ed by Jay Bailey and Karel
Luyben (University o f Technol ogy,
Delft, Netherlands). The mai n
t heme that emer ged f r om this
discussion was that engineers must
be i nvol ved t hr oughout t he dis-
cover y and devel opment o f new
bi ot echnol ogy product s and not onl y
duri ng the latter stages o f c om-
mercial pr oduct i on, as is oft en t he
case. It will be i mport ant t o train
biologists t o appreciate t he tools
t hat engi neeri ng and the physical
sciences bri ng t o bi ot echnol ogy
research.
The quality and diversity o f this
meet i ng was a clear demonst rat i on o f
how vibrant and diverse bi ochemi cal
engi neeri ng research is and h o w
rapidly it has been evol vi ng alongside
mol ecul ar bi ol ogy. The significance
o f bi ochemi cal engi neeri ng t o the
bi ot echnol ogy enterprise is expect ed
to be featured even mor e strongly
at t he next r ecombi nant DNA
conference, t o be organi zed by G.
St ephanopoul os ( MI T, Cambri dge,
MA, USA) in 1996.
References
1 Wi_ilfing, C. and Plfickthun, A (1994)
J. Mol. Biol. 242, 655
2 Bailey, J. E. (1991) Science 252, 1668
3 Robinson, A. S., Hines, V. C. and Wittrup,
K. D. (1994) Bio/Technology 12, 381
4 Mork61, T. etal. (1994) Ann. NYAcad. &i.
721,218-233
5 Lee, S. Y., Lee, K. M., Chang, H. N. and
Steinb~ichel, A. (1994) Biotechnol. Bioeng.
44, 1337-1347
George Georgiou
Department of Chemical Engineering, University
of Texas, Austin, TX 78712, USA.
TIBTECH MARCH 1995 (VOL 13)