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ProteinSynthesisin Cell-FreeReticulocyte
Lysateson Multi-HourIncubation
Mnnn A. FINDEIS
ANDGEoncE M. WnITESIDES*
Department of Chemistrg, Haruard Uniuersity,
Cambridge,MA 02138
Received
February19, 1987;AcceptedMarch23, 7987
ABSTRACT
Cell-free protein synthesis in rabbit reticulocyte lysate translation
mixtures has been studied during multi-hour incubations. In an impaired lysate obtained from cells stored at 0'C before lysis, and showing a low initial rate of synthesis, translation could be stimulated by a
factor of 4by including RNase inhibitor and additional ATP and GTP.
In translation mixtures prepared from normal lysates, protein svnthesis could be improved by -50oh by the addition of excessGTP. The
observed increases in protein synthesis were obtained by improved
maintenance of the initial rate of synthesis.
I nde x E n tri e s : C e l l -fre e p ro te i n synthesi s; cel l -freetransl ati on;
reticulocyte lysate, and behavior on multi-hour incubation.
INTRODqCTION
The in vitro, cell-free synthesis of proteins is a much more difficult
enterprise than in vivo synthesis using recombinant DNA technology,
but it is a technique of real potential utility for the synthesis of proteins
containing labeled or unnatural aminoacids, and for synthesis of proteins
containing certain types of unnatural amino acid sequences. Chemical
methods work well for the synthesis of relatively small peptides ( < 40
amino acid residues in length) but lose efficiency as synthesis of larger
products is pursued (1,2). For larger proteins (MW>20,000), chemical
synthesis may never be able to rival the combination of rDNA methods
coupled with fermentation. Becauseof our interest in the application of
* A u t h o r t o w h o m a l l c o r r e s p o n d e n c ea n d r e p r i n t r e q u e s t s s h o u l d b e a d d r e s s e d .
Applied Biochemistrg and Biotechnologg
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Fi.ndei.sand Whitesi.des
enzymes in organic syntiresis (3*5) we \,vereinterested in the suitabilitv of
cell-free enzvme-containing extracts for p'rreparativeprotein synthesis.
Reticulocvtes,immature red blood cells, are more metabolicallvactive
than ervthroivtes, mature red cells (b,7). Reticulocytesactivelv svnthesize protein, of 'ut'hichapproximateh. g}ot'ais globin that combines with
endogenous heme to form hemclglobin (8-1A1. Cell-free re'ticulocvte.
h'satescontitrue to svnthesizeprotein at - 60aiaof the rate of intact ce.lls
for up tti I l'l after cel] breakage(17) and have not been equaied in their
high level of translationaiactivitv (12).Becauseof the high relative'activitv
clfthese'lvsatesin comparisonto other cell-freesvstemsancithe abundance
of re-searchdone on and with these lysates r,r,echose the rabbit reticulocyte
lysate as the svstetmwith w,irichto begin our studies.
Although no studies of reticulocvte iysates have de.scribedthe beh a v i o r o f t h e s e l y s a t e so n m u l t i - h o u r i n c u b a t i o n s ,t h e i r p r o p e r - t i e cs i u r i n g
shori-term | - i h,1incubation haverbeen extensir.'e11,
characterizedas a
consequLrllceof studies of the biosvnthesis of proteins. The lvsate is a
c o m p l e x , m L r l t i - c o m p o n e n st v s t e m t h a t r e q u i r e - .a d e l i c a t eb a l a n c r i.r i t h e
concentrationsof a varietv tli compoLlnclsto maintain transiation(9,13,14).
A major require'ment olt the. rr.ticulocrrteh'satr. is for henrc in rlrdr-r trr
m a i n t a i n t r a n s l a t i o n a la c t i v i t v f o r m o r e t h a n - 1 0 m i n ( 1 1 , 1 5 - 1 9 ) l.i e m e '
i n h i b i t s t h e a c t i r . , a t i o no f t h e h e m e - c o n t r o l i e d t r a n s l a t i o n a l r e p r e s s o r
( t J C R )t h a i i n a c t i r z a t eisn i t i a t i o n f a c t o r e I F - 2 , l v h i c hi s n e c e s s a r vf o r t r a n s l a t i c r r(r1 0 , 2 A - 2 6 ) .
C e l l - f r e et r a n s l a t i o n s v s t e m s c o n t i n u e t o b e d e v e l o p e d ( 2 7 4 A ) a n c i
u s e d i n t h e s t u d v o f p r o t e i n b i o s v n t h e s i sa n d h a v e b e c o m e a s t a l r d a r d
tool in molecular biologv for studving messenger IINAs (mllNAs) and
. e s e s r r s t e m sl r a l ' t ' b e e n u s e d t o p r o o t h e r n u c l e i c a c i d s ( . 1 2 , 1 4 , 2 7 * 4 5T) h
duce istllableproducts, but therrhave been ope.ratedonlv ()r-r
a small scale,
j a t e r i asl ( 4 6 1 .A p a i e n t d e s c r i b e st h e p r o d u c t i o n
p r o d u c i r - t g. u d i u l a b e l e cm
of 6-liptttropin precursor protein in reticulocvtelysates,but thr. describecl
procedure is based solelv trn standard methods and is use.dto prnrduce
r a d i o l a b e l e dp r o d u c t t h a t i s i s o l a t e c bl t , i m m u n o p r e c i p i t a t r o n ( 4 i ; . I n a l l
casesthe merthodsdescribedare similar and use incubation pe.riodsof ncr
m o r e t h a n i . 5 h . W l r e r e a st h e r e t i c u l o c v t el v s a t c ' h a s b e e n d e s c r i b e ' da s
a c t i r z ei o r " i t o u r s " ( 7 0 ' ) ,w , e h a v e s e e n n o p t r b l i s h e d d o c u m e n t a t i o n o f
maintenanceof activity in theselvsatesfor greaterthan 2 h, and no discussion of the properties of these lysatesbey'ond these limited tinre periods.
Tvpical translation procedures use incubation periods of -1 h, since
lysate mixtures typicallv rapidly lose activitv after this period. The objectives of the u,ork describedin tiris paper were to investigatethe properties
of the reticulocyte lysate during multi-hour incubations to dete.rmilleif it
was possible to r,aintain high translational activity in this svstem.
\{e report here the results of studying two types of lysate's.First, we
obtained results using an "impaired" lysate of low, initial activitt (cclrresponding to an initiai rate of syntiresis of .3 pglmL-h of new protein based
on the incorporation of [3H]-leucine)(48) that displayed the typical loss of
Applied Biochemistrg and Biotechnologg
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CeIl-Fr ee Pro tein Sgnf h esis
171
activity aftc'r incrlbation at 25"C for t h. Supplementation of tiris l1'sate
witir placentai RNase inhibitor (RNasinj (49) and other compounds proionged activitv for more than I h. Second, we inrrestigatedthe response
of a "nclrmal" lvsate (initial activity corresporrdingto an initiai rate of synthesis of 8 p,g/ml-h of ne\4/prcltein svntiresis) to the' activit','-maintaining
conditions developed for therimpaired lvsater.The responseof the normai
i)'rut* was not as great as that of the impaired ivsatr..but furtirer experimentation shor,r'edmaintenanceol activit)' in tire nrlrnralI1's.rteior up to 4
h on irrclusion of excessLl'flt.
MATERIALS AND METHODS
Materials
Rabbit reticuiocvte lysate was prepared essentiallvbv tht' procedure
of Allen and Schw,eet(13) Tl-relvsate \^'aseither frczen inrnrediatelr,irr iiq u i d n i t r o g e n a n d s t o r e Ca t - 8 t l n C , o r f r a c t i o n a t e Cb v l i t e r a t u r em e t h o d s
t o o b t a i n i n X n ( 5 0 ) . F e m a k ' w h i t e r a b b i t s \ v e r e f r o m M i l l L r n r o kF a r n r s .
Amherst. N,4A" Phe.nvlhvdrazirle\ /as from Aldrich and rt,asrcLr'\'staliized
a s t h e h v d r u r c i r l o r i d ef r o m h o t e t h a n o l . P h e n v l h v d r a z i n es o l u t i o t r ( J 5 o a '
rvir,) w.asmade up immediatelv before use, neufralizeciit'itii i \ NaOii
to pl{ 7, and filtered throug}r a sterile .22 p"mfilter (Millex G3, Mil}ipore').
Carbon rnonoxide \{as fronr Matheson. l3H]-i.eucine, Econofluor, anci
Protclsolwere from NL.rt'England Nuclear. IlNasin rt'as frrlm f'ronregar
tsiotec.Additionai biochemicals\vL.rel'nrm Sigma.
TnanslationMixtures
Translation nrixtures \\'ere forrnulated based on reported procecjures
( . 1 4 , 7 6a1n d a s n o t e c li n t h e f i g u r e l e g e n d s . A i l s o l u t i o n s w e r e p r e p a r c i i
vvith distillecl deionized water" I)lastic micrcltubes and pipet tips lr'r're
handled exclusivelv rt,ith gloved hands. Fnrzen lysate rvas than'r.d bt'
brief harrd rn'arming and vortexing and then placed on ice. Translation
mixtures \^/ere made up at 0"Cl bv combining a master mix (16) w'ith
thawed lvsate. Iladiolabeled leucine (neutralized, .9 pCilpA,,59o r,,lt')
was added last followed b\. vortexing and placement in a thermclstatte'd
( + . 5 u C ) w a t e r b a t h . I n c u b a t i o n s\ , v e r ea t 2 5 o C u n l e s s c l t h e n r y i sneo t e d .
Varied Cell Washing Proceduresin the Preparationof Lysates
Fresh reticulocyte-richblood was coliected into buffere'dsaiine (134
mlvtrNaCl, 5 mM KCl, 7.5 mM MgClr, 10 mM HEPES, pI4 7.2 r,r'ithKOH)
containing 5 mM glucose.The cell suspensionwas filtered through cheesecloth and,7 x 50 mL pc-rrtionswere centrifuged for 10 min at 2500rpm (650
rpm, SS-34rotor). The plasma was decanted from the packed cells. The
cells in each tube were washed with a different solution as described in
Table 4: The packed cells were suspended in a tcltal volume of 40 mL, cenApplied Biochemistrg and Biotechnologg
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Findei.s and Whitesides
trifuged for 10 min at 2500 rpm, and the supernatant was decanted from
the packed cells; this procedure was repeated twice more.
After washing, each tube contained 5 mL of packed cells. Deoxygenated ice-cold water (7.5 mL) was added to each tube followed by vortexing
to lyse the cells. The lysis mixtures were centrifuged for 20 min at 15,000g
(11,000rprri, SS-34rotor). The lysates were divided into 1-mL and .2-mL
aliquots in plastic microtubes and immediately frozen in liquid nitrogen
and stored at -80'C. Each sample yielded 9 mL of lysate. Both lysates
required little adjustment of the Mg2+ level; .25 mM GTP was added for
optimal activity. Lysates 1-3 were optimally supplemented with 20 pM
hemin. Lysates 4-7 did not need added hemin but 10 plvl was included
to ensure an adequate level. Lysates 1.-7 were tested for activity over 3 h
incubations. Master mix was made up and used directly for lysates 1-3.
The remainder was held under nitrogen for 10 min before combining with
Iysates 4-7.
Sucrose Gradient Analysis (51')
Sucrose solutions were prepared in 10 mM TRIS-HCI, pH 7.4 containing 2 mM magnesium acetate,50 mM KCl, and .7 glL gelatin (61) Lysate
samples (60 pL) were diluted with 2 volumes of gradient buffer and applied to linear gradients (12 mL, I5-30o/osucrose w/v). Gradients were
centrifuged in the 70.1 Ti rotor at 45,000rpm for 30 min and allowed to
stop with the brake off . The gradients were passed through the UV cell of
an LKB Uvicord II detector equipped with a chart recorder and operating
at 254 nm.
Sample Preparation for Liquid Scintillation
Countin g (14)
Aliquots (5-10 pL) were removed from an incubation mixture with a
Hamilton syringe (#801N 10 pL) and blown into .5 mL of cold water in a
12x75 mm disposable culture tube. The syringe barrel was rinsed 4 or 5
times to ensure complete transfer of the sample and to clean the syringe
for the next aliquot. Sampleswere then stored at 4oC until a set from an
experiment were ready for further processingtogether. NaOH (1N, .5 mL)
and 30o/ohydrogen peroxide (50 pL) were added to each tube followed by
heating at 37 oC for 15 min or standing at 25"C for 20-30 min. Ice-cold trichloroacetic acid (25o/ow/v) was added to each sample, and the samples
kept at 4oC for th. The contents of each tube were transferredonto a 2.1
cm CF/C glass fiber filter. The sample tube was rinsed twice with 1 mL of
8oloTCA and these washes were passed through the filtered precipitate.
The filter was placed in a glass LSC vial so that it was lying flat on the bottom of the vial and 1mL of ethanol/Protosol(1:1) was added. The sealed
vial was heated at 55-60oCfor 30 min and ethanol (.5 mL) containingl0o/o
glacial acetic acid was added followed by 10 mL of Econofluor. Radioactivity was measured in the scintillation counter and reported as the observed counts per minute.
Applied Biochemistrg and Biotechnolog g
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Cell -Fr ee Pro tein Sgnthesis
173
RESULTS
Impaired Lysate
into acid precipitatable
The kinetics of incorporationof 1ap1,-teucine
in the impairedlysateis shown in Fig. 1. The lysate
protein (73,79,52,53)
showed a near linearrateof synthesisfor L h, and then quickly lost activity. Supplementingthis lysatewith eitherRNasinor sRNA resultedin improved maintenanceof the initial rate of incorporationand greateroverall
protein synthesis(Fig. 1). In combinationtherewas no additionalbenefit,
and excesssRNA proved to be inhibitory. Incorporationin the supplemented lysate was maintainedat nearly the initial rate for 2 h and was
15000
10000
tr
CL
o
5000
0
T
I
I
0
2
4
6
B
10
r (h)
Fig. 7. Incorporation of [3H]-leucine into acid precipitated material in
impaired lysate translation mixtures: unsupplemented, (l); with 1,tJ IpL added
RNas in, ( t r ) .
Applied Bio chemistrg and Biotechnologg
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Findeis and Whi.testdes
doubled again in the next 5 h (Fig. 1). Subsequent experiments showed
that on storage the sRNA preparations tended to become inhibitory. In
general, becauseof the equivalent activating ability of RNasin, it was used
alone without sRNA.
Addition of dithiothreitol (DTT) (54,55) produced modest gains in
incorporation but high levels were inhibitory (Table 1). Addition of extra
creatine phosphate and creatine kinase afforded no benefit, nor did the
addition of 1,10-phenanthroline, a known inhibitor of proteasesin reticulocyte lysates (56).Extra ATP (1 mM), GTP (.2 mM), and Mgz+ (1 mM)
(57,58) resulted in a modest increase in incorporation in an 8 h incubation (Fig. 2). Clucose-6-phosphate(.4 mM) was also modestly activating
( 1 2 , 5 5 , 5 9 , 6 0. )
Addition of the protease inactivator phenylmethylsulfonylfluoride
(PMSF) resulted in no effect on incorporation. A control experiment to
evaluate the effect of ethanol, which was used as a cosolvent for PMSF,
surprisingly showed activation of the lysate. Others have reported that
).3o/o v/v of ethanol inhibits protein synthesis in reticulocyte lysates
(61,62).We found ethanol at up to .2o/oto be activating (Figs. 2, 3).
The results obtained in maintaining the activity of the impaired lysate
are significant in comparison to the behavior of the unsupplemented translation mixtures. Even in the optimized translation mixture, however, the
overall level of incorporation corresponds only to a level of protein synthesis of - 2 p"glmL. which is about 2o/oof the activity of the most active
lysates reported (12).
Normal Lysate
This lysate displayed high initial activity for 2 h, after which activity
was rapidly lost (Fig. 4). Adding extra ATP, GTP, and Mg2+ was modestly
activating (Fig. 4). Increasing the temperature of incubation from 25oC to
30'C approximately doubled the initial rate, but shutdown of incorporation occurred at the same level of incorporation (Fig. 5).
An interesting observation came from passing carbon monoxide over
a translation mixture prior to incubation. Only one-fourth of the incorporation of the control was obtained in 4 h by which time the control was
Table 1
Effects of Increasing Amounts of DTT on Incorporation in
Impaired Lysate Incubation Mixtureso
Experiment
Control
DTT, 25 mM
Cpm, 2 h
1pL
2pL
5pL
6996
8345
6522
6174
Cpm, 3 h
9524
8779
10773
11350
' Reaction volume, 50 pL; reaction time 3 h; 10 pL aliquots.
Applied Biochemistry and Biotechnologg
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Cell -Free Pro tein Synfhesfs
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20000
15000
A
A
s
tr1
cL'
o
I
.'r
./
:/
0000
,/tr
l/
u
/
5000
0
2
4
6
B
10
12
r (h)
Fig. 2. Translationin the impaired lysate system:(a) control (rl); (b)
with .2vlv ethanol(f); (c)controlsupplementedwith 1 rnM ATP, .2mM GTP,
and 1 mM MgCl, (A).
shut down. In the following2h after being opened to air, the level of incorporation increasedto near that of the control (Fig. 6).
Concentration of normal lysate by ultrafiltration increasesthe "specific activity" of translation mixtures, but not by as much as the lysate is
concentrated (63) Diluted lvsate (10o/a
r'/v in a translation mixture) yielded
no activity, presumablv becauseof dilution of components of the lysate to
less than optimal concentrations.
As translation proceeds in lysates, creatine phosphate is consumed
and inorganic phosphate is released.To evaluate the extent to which free
phosphate might inhibit translation mixtures, 0-20 mM sodium phosphate
was added to the lysate. Increasingphosphate concentrationproduced a
Ap p lie d Bio c hem is t rg an d B io te chno lo g g
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Findets and Whitesides
60000
r1
40000
./
tr
CL
o
20000
0
0.00
0.05
0.10
0.15
0.20
EtOH
Fig. 3. Incorporationin the impairedlysatesystemaftert h with varying
amountsof added ethanol.
near linear decreasein the incorporation observed in a 2-h period (Fig.7),
likely in part a result of complexation of Mgz+. In lysates containing 20 mM
added phosphate, 2 mM added Mgz+ resulted in recovery of half of the
initial activity.
Pyruvate kinase and phosphoenolpyruvate are a less effective phosphorylation system in whole reticulocyte lysate than creatine phosphate/
creatine kinase (11). Whereas creatine kinase does not accept GDP as a
substrate (64), GTP is reported not to be depleted in t h incubations (20).
To test if GTP might be a factor in the loss of translational activity, several
different combinations of phosphorylating potential were tried (Table 2).
Consistent with previous results, mixtures containing PEP were inhibited
(11). No activation was achieved by including PEP/PyK in incubations
along with CrP/CrK. PyK alone had no effect on incorporation. Added
Applted Bio chemistrg and Biotechnolog g
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Cell -Free Protein Sgnthesis
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40000
tr
30000
--- - t - -
-j--tr
E
o.
o
20000
It
10000
0
2
4
B
6
10
r (h)
Fig. 4. Translational activity in the normal lysate system over 10 h: (a)
standard translation mixture (I); (b) supplemented with 7mM ATP, .2mM CTP,
a nd 1 m M M gC l , (f).
Table 2
Effect in Normal Lysates of Different Phosphorylating Systemsd
cP^
Experiment\ Addition
CrK + CrP
L, control
2
3
4
5
+
+
PyK
PEP
@1h
@ 2h
+
+
+
+
+
37518
12339
20139
77713
35418
46825
1321.1
29555
1.4507
42562
+
o CrK and CrP were the usual amounts if present, PyK (.5 mg/ml-) and PEP (.7 M) were
added at 4o/o(v/v).
Applied Biochemi.strg and Biotechnologg
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178
Findeis and Whitestdes
50000
40000
|
tr-
--tr
l/
It
,i/
30000
E
o.
o
20000
10000
0
2
3
4
r (h)
F ig. 5.
(b)30'c (t).
In c u b a ti o n o f n o rma l l v s ate transl ati on mi xtures at (a) 25' C { i -l );
Mgz+ brought the activity in the combined system up to the control level
and indicated that PEP reduced Mgz+ activity (Table 3).
CTP is absolutely required for translation (6,8). Its concentration in
lysatesis about .1mM (14). GTP is also capableof substituting for hemin
in lysates as a blocker of the activation of HCR in preincubations in the
absence of an energy system (20). We found that adding GTP-Mg2+ resulted in increasedincorporation in 2 h incubations (Fig.S). While shutdown of translation was siowed and incorporation significantly increased,
the system was still nearly stopped after 2 h.
Maintenance of high activity in the reticulocyte requires the availability
of either chemical or biochemical reducing agents. DTT and thioredoxin
have been shown to be useful sources of reduction potential in iysates
(59,65), one or the other required in conjunction with compounds that
allow the lysate to generateNADPH such as glucose-6-phosphate.Lysates
Ap p iied Bio c he m is trg and Bio techno Iog g
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50000
40000
EI-____
r-oo
30000
E
o.
o
20000
10000
0
23
4
5
6
T (h)
Fig. 6. Incorporation in normal lysates at 25oC showing the effect of incubation under carbon monoxide for 4 h (l) before exposure to air; control, no
co (!).
Table3
Effectof Added MgCl, on trncorporation
in Norrnal LvsateMixtures
ContainingPyruvateKinase(20 pgiml) and PEP(4 mM),'
Experiment
CP*
control, no PyK or PEP
w/PyK, PEP
no added MgCl,
1 mM MgCl,
2 mM MgCl,
4 rnM MgCl,
5 mM MgCl,
14158
7452
74748
10952
8386
5062
o Incubations were for 2 h.
Applied Biochemistrgand Biotechnologg
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F indei.sand Whitesides
20000
15000
El
cL' 0000
CJ
5000
0
5
10
15
20
Phosphate(mM)
F ig. 7. E ffe c t o f a d d e d p h o s p h a te (.2 M sodi um phosphate, pH 7 .2) on
incorporation in 2 h incubations of normal lysate translation mixtures.
are very sensitive to oxidized glutathione (GSSG), and addition of GSH
can lead to inhibition because of small amounts of contaminating GSSG
(66,67). There has been a caution about adding thiols to translation mixtures (66). Under certain conditions, however, DTT has been found to be
essential for maintaining activity in lysates (67).GSH does not function
as well as DTT as a reducing agent. In normal lysates we found DTT to be
inhibitory, in contrast to the data from the impaired lysate where DTT
was an important element of the improved translation mixture to insure
stability of added RNasin.
An alternative to addition of thiols to maintain reduction potential in
the lysate is to maintain the NADPH concentration. Added NADPH is
not very efficient in this regard compared to the use of compounds to
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25000
20000
E
15000
CL
o
10000
5000
GTP-Mg2+(mM)
Fig. 8. Effectof added GTP-Mg2*on incorporationin 2-h incubationsof
normal lysatemixtures.
allow regeneration of NADPH from NADP; excessNADP can inhibitively
disturb the NADPH/NADP ratio (55). In partially fractionated lysates, .4
mM G6P is effective for maintaining NADPH (55,59). We found that
added G6P over 1 mM was inhibitory, more so than the same amount of
inorganic phosphate. Adding MgCl, did not offset this inhibition. Addition of glucose, which can function as G6P after phosphorylation by hexokinase, was inhibitory, as was NADP. This observation contrastswith the
reported activating effects of both glucose and NADP in shorter incubations (66).Glutathione reductasewas included in lysateswith and without G6P and NADPH, and no advantage was observed, consistent with
previous observations that GSH is not oxidized to an appreciableextent
during incubations (66,67). The lysate pH optimum for translation has
Applied Biochemistrg and Biotechnolog g
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Ftndeis and Whitesides
been established as narrow at pH 7.2 (77,59). We found that lysate pH
during incubation is constant, varying less than .1 pH units after 2 h and
loss of activity.
Enzymic oxidation of G6P to recycle NADPH could ensure availability
of NADPH if otherwise limited bv enzymes lost in the preparation of the
lysate (68). Inclusion of sufficient G6P-dehydrogenase to convert the
available G6P to form NADPH had no effect on incorporation. Previous
reports on different types of impaired lysates found that adenosine,
cAMP, and related compounds could function as activators of translation
(65,67,69).We found in the normal lysate that cAMP was verv inhibitor,v.
Aged lysates deficient in essential factors for translation are reported
to regain full activity on addition of partial volumes of lysate or ribosomefree lysate supernatant (16,66,67,70).
Our normal lysate, after having lost
activity on extended incubation, showed no further activity on the addition of fresh lysate to inactivated translation mixtures.
To probe the effect of oxvgen on cells before and during lysis, a series
of lysates were prepared from the same batch of cells using differentl,v
prepared cell-washing soiutions (Table 4). All of these specially prepared
lysates were similar in translational activitv. Slightly higher activities
were actually obtained from those cells r.r'ashedwith solutions that were
not deoxygenated nor supplemented with hemin. Less active lvsates
have been obtained previously from cells incubated extensivelv under
anaercrbicconditions at elevated temperatures (65,66). The period required to wash cells several times (7-2 h), even though at 4"C, mav be
sufficient to lead to impaired lvsates. As in previous experiments without
extra CTP, incorporation proceeded weli for t h and then was essentially
stopped.
Sucrose gradient analysis of polysome, ribosome, and subunit aggregation has been extensivelyused in other studies of reticulocytelysates
(11,19,22,23,55,65-67,69,71).
In studies on lysatesthat have been impaired
Table 4
Formulation of Cell-Washing Solutions and Extent of Incorporation in
Translation Mixtures Made with Lysates from These Cell Preparationsd
No.
Washing Solution
CP-
1
2
Sa l i n e (1 ,3 4mM N a C l , 5 m M K C L, 7.5 mM MgC l r)
S a l i n e , b u ffe re d (1 0 mM H EP E S , pH 7 .2 w /K OH ,
5 mM glucose)
Saline, buffered, deoxygenated
Saline, buffered , w I 20 pM hemin
Saline, buffered, w/ hemin, .5 mM adenine
Saline, buffered, w/ hemin, 1 mM DTT
Saline, buffered, w/ hemin,
containing 2-mercaptopropanol
41680
3
4
5
6
7
4770A
3824A
3n7A
34945
3$44
30990
o Incubations were for 3 h.
Applied Biochemistrgand Biotechnology
V o l .1 5 , 1 9 8 7
CeII-FreeProtein Synfhesis
183
in a manner to cause loss of activity within several minutes, the general
observation has been that active lysates show high molecular weight aggregates corresponding to polysomes that disaggregateas activity is lost
resulting in the accumulation of lower molecular weight ribosomes and
ribosome subunits. This general pattern is observed in instances where
shutdown is believed to be caused by loss of initiation factors. In our normal lysates, after 2h at 30oCthe high molecular weight peaks are diminished in size and lower molecular weight peaks are larger indicating a state
of disaggregation similar to that in lysates where initiation is inhibited
(Fig.e).
+I
I
I
I
t z54
D e c r e o s i n gD e n s i t y Fig. 9. Polysome profile in a 15-30o/osuCrosegradient: (A) translation
mixture applied to the gradient after 5 min at 30'C; (B) translation mixture applied to the gradient after 2 h at 30"C. Gradients were centrifuged for 30 min at
4 5, 000r pm in t h e 7 0 .1 T i ro to r.
Applied Biochemistrg and Biotechnologg
V o l .1 5 , 1 9 8 7
184
Findei.s and Whitesides
When an incubation mixture rvas held under a stream of nitrogen gas
for 10 min at 0"C before starting incubation at 30oC it showed20o/ogreater
incorporation over that in lysate not treated with nitrogen. If the lysate
was subjected to a degassing procedure with 2 freeze-thaw cycles under
nitrogen and vacuum, the resulting lysate had nearly no activity.
DISCUSSION
lmpaired Lysate
In optimizing the activity of cell-free translation in the reticulocyte
lysate, gains in activity are only seen as a result of maintaining the initial
rate of activity for a longer period of time. A higher temperature of incubation does result in an increasedinitial rate of synthesis but the resulting
overall level of synthesis is the same as at lower temperature (72). The impaired lysate results are of limited use as they apply to a system with low
overall activity. It is interesting, however, that translational activity in the
impaired lysate can be maintained for several hours in this system. Still,
with sustained activity the level of newly synthesized protein achieved in
10 h is far less than that achieved by normal lysates even with their much
shorter period of sustained activity. A previous report also describeslow
activity lysates that maintained activity longer than usual, with lower
overall synthesis (13).
A major limiting factor in the impaired lysate is certainly the loss of
tRNA soon after the start of an incubation that can be offset by the addition of extra tRNA or RNasin. Addition of other compounds to the translation mixtures provided little further activation of protein synthesis.
The metabolic activity of red cells is significant, even at 0oC (73). Even
though stored overnight at 0oC, the cells from which the impaired lysate
was made may have become depleted in ATP. Such ATP depletion has
been shown to cause inhibitio.t oi t.utlslation and a similar effect seems to
have occurred in our impaired lysate.
Normal Lysate
Normal lysates do not require added RNasin or sRNA and do not exhibit enhanced translational activity under the conditions with which the
impaired lysate does. The inhibitory effects of DTT in the normal lysate
contrasts with activation by DTT in the impaired lysate. This observation
indicates that whereas the impaired lysate has reduced biochemical reducing power, the normal lysate is still effective in maintaining reducing
power. In normal lysates additional DTT affords no advantage, and in
high quantities is inhibitory. In contrast to the impaired lysate, normal
lysates are not activated by low levels of ethanol.
The effects of increasing temperature suggests that in a given lysate a
certain amount of "metabolic activity" will lead to inactivation (11,65).
Applied Biochemistrg
and Biotechnologg
V o l .1 5 , 1 9 8 7
Cell -Free Protein Synthesis
185
This suggestion is consistent with the result from incubation of the normal lysate under carbon monoxide. This incubation displayed inhibited
translation, but exposure to air restored the lysate to the same level of
activity as was observed for a normal incubation. Carbon monoxide apparently binds reversibly to components of the translation mixture to
"freeze" the activity of the lysate. This inhibition is not related to a specific requirement by the lysate for oxygen since the lysate is modestly
more active when incubated under nitrogen than when incubated under
air. This result contrasts with the properties of lysates obtained from cells
maintained under nitrogen, conditions that cause the cells to become
energy starved and that result in inhibited translation in the resulting
lysates (65).
Although the lysate is highly sensitive to the effective concentration
of Mgz+, inorganic phosphate is not sufficiently inhibitory to account for
the loss of translational activity. A.y partial inhibition by phosphate is
ruled out by experiments in which small amounts of Mgz* were added
during incubation to offset accumulating phosphate and no activation
was observed. The binding of creatine phosphate with Mgz+ is apparently
competitive with the binding of inorganic phosphate. Thus, the effect on
translational activity of accumulating inorganic phosphate from the consumption of creatine phosphate is, in effect, "buffered." In contrast, PEP
has a high affinity for Mg2* and this tight binding can have a deleterious
effect in the lysate. As PEP is consumed the activity of Mgz+ increasesand
becomes partially inhibitory, although never sufficiently inhibitory enough
to stop activity completely.
Loss of activity in these translation mixtures has been demonstrated
in lysates in which HCR cannot be detected (67). Since HCR is catalytic
and can inactivate a 100-to 1000-foldmolar quantity of ribosomes, an undetectablelevel of the inhibitor may be sufficient to lead to loss of translational activity after 2 h. As eIF-2 is depleted the lysate may not become
dependent on the concentration of this initiation factor until its concentration is so low as to result in rapid loss of activity.
The most active lysatesreported synthesize new protein with an initial
rate of synthesis of - 100 mg/L-h (12). Our normal lysates consistently
had initial activities of 8 mg/L-h and produced a total of 20 pglpL of new
protein. Keeping in mind the high concentration of protein already present in the reticulocyte lysate (>50 glL, mostly hemoglobin), it becomes
apparent that isolation of a translation product could be very difficult
without a selective method for removal of the product from the translation mixture, e .8., immunoprecipitation or affinity binding.
CONCLUSIONS
Protein synthesis in this cell-free system is not currently suitable for
preparation of gram quantities of product. Even if a sufficient quantity of
Applied Biochemistrg and Biotechnologg
VoI.15. 1987
186
Findets and Whitesides
the appropriate mRNA were available, which is feasablewith the recent
development of RNA splicing methods (74), only a fraction of a gram of
product could be expected to be obtained from a liter-sizecitranslation
mixture that would require - 15 rabbits. For small-scalepreparative applications, the reticulocyte has, however, potential useful application.
A cell-freetranslation system offers opportunities for the manipulation
of protein structure not practicalbv fermentation or chemicalsynthesis.A
ceil-free system can be more efficient in the incorporation of added amino
acids, important if an amino acid labeled with a stable isotope were being
introduced into a protein. If it were desired to introduce an unnatural
amino acid, cell-free synthesis would offer a potential route by using
chemically synthesized aminoacyl-tRNA (75) containing the unnatural
amino acid residue that would be accepted by the translational svstem
and incorporated into the protein. Whereas site-specificmutagenesiscan
introduce an alternative natural amino acid, a fermentive svstem cannot
usuallv be expected to accept an unnatural amino acid, especiallvif its
structure is significantly different from natural amino acids.
Small-scalecell-free protein synthesis has the potential to be important as one approach to the svnthesisof unnatural proteins. Although the
reticulocyte lysate translation system has its limitations, it is considered
the mosf active system for the iranslation of eukaryotic mRNA's (33). It
may be possible to improve on the activity and cost of current cell-free
translation systems by investigating systems derived from cultured celllines. The limited development and current usefulness of such svstems
has been discussedbriefly (33).
ACKNOWLEDGMENTS
This work was supported by the National Institutes of Health, Grant
GM 30367.MAF was the recipient of an Exxon Foundation Fellowship at
Harvard during 7982-7983.
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