Biochimie (I 991) 73, 983-989 © Soci6t6 fran~:aise de biochimie et biologie mol6culaire / Elsevier, Paris

983

Superexpression and fast purification of E coli initiation factor IF2 KK Mortensenl, NR Nyengaardl, JWB Hershey2, S Laalami3, HU Sperling-Petersenl*

ILaboramry of Biodesign, Department of Chemistry, Aarhus University, DK-8000 Aarhus. Denmark; 2Department of Biological Chemistry. University of California, Davis CA 956 i 6, USA; .~lnstitut de Biologie Physit'o-chimique. 13. rue Pierre et Marie Curie. F-75005 Paris, France (Received 31 January 1991; accepted 8 February 1991 )

Summary - - For the production of large quantities of E colt initiation factor IF2 we have constructed an improved overexpression system. The gene infB was cloned into the thermo-inducible runaway plasmid pCP40 [I ] and subsequently transformed into the E colt strain C600[pci857]. In this system the expression of infB is under the control of the strong promoter ~PL and the cells carry the plasmid pc1857, which contains a thermosensible kcl repressor. Overexpression of IF2, which is approximatley 30 times higher than the expression in wild-type-ceUs, is induced at 42°C and continues for 2 h at 37°C. From these cells pure and active IF2 was obtained using a novel 3-step FPLC-procedure consisting of ion-exchange liquid chromatography on Q-sepharose HP, MonoQ and MonoS. In approximately 8 h, 5 mg of pure and active IF2 can be obtained from !0 g overproducing cells. This corresponds to 5 mg of IF2 per litre of medium. The purification was monitored by Western immunoblotting and the activity of the purified factor was tested by measuring the stimulation of binding of the initiator fMet-tRNAMet to 70S ribosomes in the presence of GTP and poly(A,U,G) as messenger RNA. Compared with previous methods our purification procedure avoids the use of materials such as DEAE-cellulose and phosphocellulose which have relatively poor flow rates. In addition to the higher flow capacity of Q-sephaios¢ HP, this new matrix can be loaded with an $30 supernatant. This avoids the need for the preparation of a ribosome-free S 100 supernatant by ultracentrifugation and thus the need to keep IF2 in solution with membrane and periplasmatic proteases for at least 2-3 h. By this procedure, only minor amounts of the proteolytic 65 kDa fragment IF2), are formed during the purification of IF2. Stability tests showed that IF2 is extremely labile in the $30 supernatant. However, the purified IF2 is not an unstable protein as previously believed.

superexpression / recombinant IF2 / initiation factor IF2 / protein purification Introduction The initiation of protein synthesis in Escherichia colt is promoted by three proteins, initiation factors I F l , IF2 and IF3 (for an extensive review, see [2]). IF2 is the largest of the initiation factors and is present in bacterial cells in two size classes, IF2tx (97.3 kDa) and IF2[3 (79.7 kDa) [3, 4]. It interacts with at least three components of the initiation pathway: GTP; fMet-tRNAf~et and ribosomes. Through these interactions, it promotes the binding of fMet-tRNAfMet to the 30S ribosomal subunit, and catalyses the hydrolysis of GTP following 70S initiation complex formation [5]. The gene for IF2, infB, has been cloned and mapped [6] and the sequence of IF2 was deduced from the sequence of its gene [7]. A six-domain structural model for IF2 has been proposed [8] and a more detailed model for domain IV, the G-domain, has

been suggested, based on X-ray crystallographic data from the homologous elongation factor E F - T - [0] In order to test our model with crystallographic data on IF2, large quantities of ultrapure protein are required. The purification procedures for the E colt IF2 described previously lasted 1-3 weeks and resulted in a loss of native IF2 due to partial degradation [ 10, I I ]. The major fragment, IF2~, (65 kDa), appears to result from proteolysis of IF2, since it was not detected in fresh extracts by immunoblotting with anti-IF2ot [3, 12]. The problem of this loss during purification was partly overcome by the introduction of an E colt system for the overexpression of IF2 [ l l ]. In this paper, we describe the construction of an improved overexpression system for tF2 and a new, faster purification procedure for this initiation factor. M a t e r i a l s and m e t h o d s

*Correspondence and reprints

Plasmid pCP40 and E colt strain C600[pci857] were a kind gift from Drs W Fiers and E Remaut. Gent. Belgium.

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KK Mortensen et al

Biochemical compounds were obtained as follows: restriction enzymes, T4 DNA ligase, RF Ml3mpl9, E coil strain JMI05, tRNAMet and poly(A,G,U) were from Boehringer Mannheim, Germany. Folinic acid, Tris[hydroxymethyllaminomethane (Tris) and N-[2-hydroxyethyl]piperazine-N'-[2ethanesulphonic acidl (HEPES) were from Sigma, USA. Acrylamide, N,N'-methylene-bis-acrylamide and N,N,N',N'tetraethylenediamine (TEMED), were from Bio-Rad, USA and all other chemicals were of analytical grade obtained from Merck, Germany. Buffer H: 50 mM HEPES pH 7.5; 10 mM MgCI.,; 7 mM 2mercaptoethanol. When buffer H was used with various concentrations of KCI, the millimolar concentration of KCI is shown in parentheses; eg H(100): [KCll = 100 raM. Disintegration buffer: 50 mM Tris-HC! pH 6.8; 2 mM Na, EDTA; 1% SDS: 140 mM 2-mercaptoethanol; 10% glycerol; 0.1% hromphenol blue. Q-Sepharose high performence, MonoQ high resolution 5/5 and MonoS high resolution 5/5 were from Pharmacia-LKB (Uppsala, Sweden) (Q- = quartenary amine and S- = sulfonate). [3Hl-L-methionine was from Amersham, UK. tRNA~ et was aminoacylated with methionine and formylated as described [131. The specific radioactivity was 850 cmp/pmoi tRNA.

Cell lysis

DNA-methodology

Q-Sepharose HP was treated and packed into Pharmacia High Resolution columns according to the suppliers' instructions. Pre-packcd MonoQ HR 5/5 were treated as directed by the manufacturer. All column chromatography was carried out on an FPLC (fast protein liquid-chromatography, a registred trade mark of Pharmacia-LKB, Uppsala, Sweden) setup, where all protein handling was done in a cold cabinet (Hekama) 0--4°C. Absorbance at 280 nm was determined on-line with an integrated spectrophotometer (Pharmacia-LKB UV-M Monitor, 5 mm cuvette). Details of column size and elution conditions are given under Results. All chromatography runs were followed by an SDS-PAGE of a window of the fractions containing IF2.

Construction of M 13mpl9infB and pCP40infB is described in the text and all methods employed are given in the enzymesuppliers' recommendations and in [141. Dideoxy-DNA sequencing was carried out using the Sequenase Version 2.0 kit from United States Biochemical Corporation, USA.

Cell growth The overproducing strain C600[pclssT; pCP40h~B] was grown at all times (except during overexpression) at 28°C. 1 i of LBmedium containing 100 ~tg/ml ampicillin and 50 lag/ml kanamycin in a 3-1 notched Erlenmeyer-flask was inoculated with 10 ml stationary phase C600[pcls.~7; pCP40#~B] grown in the same medium 16 h, The inoculum was started from a fresh overnight LB-agar plate containing 100 lag/ml ampicillin and 50 [ag/ml kanamycin. The flask was incubated in an orbital shaker t:~lew Brunswick Scientifik G25) at 250 rpm. After about 7.5 h the culture had a cell density of As~0= 1,0 (Pharmacia-LKB Ultrospec II spectrophotometer). The flask was transferred to a 42°C waterbath; here the heat transfer was very effective - the temperature in the culture reached 42°C in less than 5 min. After 20 min the flask was transferred to a 37°C orbital shaker at 250 rpm. After 2 h incubation the culture had reached a cell density of Ass0 = 3.24, and the flask was cooled rapidly in an ice-water bath and the cells were harvested in 500 ml flasks by centrifugation 10 min at 8000 rpm (4°C) in a Sorvall GSA rotor, The cells were washed in buffer H(100) and collected by centrifugation 10 min at 8000 rpm (4°C). Cell dry weight was determined after baking wet cells at 80°C for 24 h. To monitor the cellular protein biosynthesis and for the control of overexpression 1 ml aliquots of the culture were taken at different times during the incubation. The samples were pelleted at 14 000 g for 5 rain in a Hereaus BiofugeA centrifuge and resuspended in one voi of disintegration buffer ensuring an equal concentration of cells in all samples. After 3 min at 100°C, all cell debris was pelleted at 14 000 g for 5 min and 20 ~tl of the supernatant were loaded onto a 10% SDS-PAGE and electrophoresed.

10 g of cells were resuspended in 40 ml buffer H(400) and passed once through an ice-cold French pressure cell (Aminco) at 1800 psi. A crystal of bovine pancreatic DNase was added to the viscous extract and the mixture was stirred on an ice-water bath for 30 min. The extract was then centrifuged for 1 h at 30 000 g (4°C) in a Sorvall SS-34 rotor. The supematant, $30, was used directly in the following purification procedure.

SDS-PAGE 10% discontinuous sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out in a Bio-Rad Protean II gel apparatus as described [ 151. The gel was stained for ! h in 0.2% Coomassie brilliant blue R in 50% ethanol/5% acetic acid and destained in the same solution without the dye.

Immunobiotting lmmunoblotting was carried out using an Ancos Semi-dry blotting apparatus as described in [I 21.

Column chromatography

N-tt,rminal amino-acid sequencing The N-terminal amino-acid sequence was determined on an Applied Biosystems 470A gas-phase micro sequenator as described in 191.

Assays Initiation factor IF2-dependent binding of formylmethionyltRNArMet to 70S ribosomes was used to determine the activity of the purified factor. The assay was carried out as described in [131. For direct protein quantification a modified Bradford Coomassie blue dye assay was employed [ 161.

Results a n d d i s c u s s i o n

Construction o f the superexpression system for infB The gene c o d i n g for IF2, inJB, w a s e x c i s e d f r o m pB 181 [ 17] as a HindllI-BgllI f r a g m e n t a n d s u b c l o n e d into the HindllI-BamHI sites in the p o l y l i n k e r o f M 1 3 m p l 9 . T h e insertion w a s c o n f i r m e d b y d i d e o x y s e q u e n c i n g a n d the c o n s t r u c t w a s n a m e d M l 3 m p l 9 infB. S i t e - d i r e c t e d m u t a g e n e s i s o n infB is presently being p e r f o r m e d o n this template.

Superexpression and purification of E coil IF2 Hi

985

RI

Hindlll, EcoRI

Hindili

EcoRl

EcoRl

infB

E.B.H.X.P

Hindlll, I~¢oRI ,,.--

Hindlll

EcoR1

I Amp r

~PL

Fig 1. The construction of the expression vector pCP40infB for E

coli initiation factor IF2. The restriction endonucleas¢ sites in the polylinker of pCP40 are abbreviated as follows: E = EcoRI; B = BamHl; H - HindllI; X = Xbal; and P = Pstl. Ampr indicates ampicillin resistance and ori(ts) is the thermosensitive origin of replication of pCP40.

The D N A fragment containing infB was then excised from the two unique restriction sites in M I 3, H i n d l l l and E c o R l , and ligated into the runaway expression vector pCP40 [1], as shown schematically in figure 1. In the recombinant vector, pCP40infB, the infB gene is under the control of the ~L-PL promotor. This plasmid was transformed into the E coli strain C600[pcIss7]. In this system (C600[pcIs57; pCP40infB]) the

expression of IF2 is controlled by the temperaturesensitive ~,-pcIs57 repressor. At 28°C, below the permissive temperature, only few plasmids are present in the cell and at the same time the ;L-PL promotor is repressed. Above the permissive ten~perature, the plasmid copy number increases to about 1000 per cell and, as a result of the inactivation of the repressor, the plasmids are transcribed and infB is vigorously expressed. The cells carrying the plasmid pCP40in/B (C600[pcIs57; pCP40infB]) were grown at 28°C to A~0 = I as described in Materials and methods. In order to induce replication of pCP4OinfB and expression of infB, the temperature was then raised rapidly to 42°C and kept constant for 20 min. The culture was then incubated at 37°C during the subsequent growth with high expression of IF2. 2 h growth at 37°C resulted in A55o = 3.24, equivalent to 10 g of cell wet weight or 1.8 g of cell dry weight per litre of medium. Figure 2 shows the growth curve and an SDSPAGE analysis of samples of lysed cells before and after induction. As seen in lanes 8-11 only IF2 is strongly overexpressed. The induced C600[pcls57; pCP40in~B] system expresses IF2 to approximately 35% of

Ass0

minutes MR 9&K --='

3.0.

~

67K -~ 43K ~,

30 K ~-

2.0

1.0

28°C

37°C

0.0 0

200

400

min.

600

800

1000

Fig 2. Growth curve for E coli cells strain C600[pc1~57; pCP40infB] overproducing recombinant IF2. The time of induction is indicated together with temperature changes. At different intervals, samples were taken from the incubation medium and the protein contents of the cells were analyzed by SDS-PAGE. The results are seen on the inserted Coomassie blue stained gel where each lal=e contains the extract from 500 l,tg cells.

986

KK Mortensen et ai

A28o 2.0-

S301,=--------Iro¢llons

~0-58 . . . . . . . . . . .

"~

KCt,mM

DEAE-cellulose, phosphoceUulose and hydroxylapatite as well as size-exclusion chromatography on different gel filtration matrices. These are all materials with relatively poor flow rates. These kinds of material are not employed in the procedure presented here. Instead, we have designed an FPLC procedure using the anion-exchangers Q-Sepharose High Performance and Mono Q followed by a final purification step using the cation exchanger Mono S.

Q-Sepharose HP and MonoQ

1.0. &00

I

300 200

,,, f 1000 ............

L6'"~s

~;~"

"

FRACTION NO.

Fig 3. Chromatography on Q-Sepharose HP of $30. Column dimensions: 1.6 x ! 0 cm = 20 ml Q-Sepharose HP; sample: 50 mi $30 containing 150 mg proteins including ribosomes; flow rate: 2 ml/min; fraction size: 2 ml. The fractions which were pooled are indicated as the hatched area. Insert: SDS-PAGE analysis of the $30 sample applied to the column and of fraction 40-58. the soluble cytoplasmatic proteins. We are proposing the term superexpression for a recombinant E coli system in which the expression of a naturally-occurring protein is boosted to a level more than 25 times higher than the level of the wild-type. Our experience has shown that continuation of growth at 42°C often results in denaturation and precipitation of the overexpressed protein. Decreasing the temperature only by a few degrees centigrade results in soluble ~'ad active gene product. The cells were opened by using a French pressure cell, and the extract was incubated for 30 min at 0°C in the presence of DNase. An $30 supernatant was isolated from the cell extract. The use of the French pressure cell to open the bacteria reduces the processing time from 1 h (the usual grinding with alumina) to a t~w minutes.

Purification of IF2 Since the discovery of initiation factor IF2 in 1966, the liquid-chromatographic methods used to purify the factor have included adsorption chromatography on

The first step in the protein purification is ion-exchange chromatography on the anion-exchanger QSepharose High performance. This matrix allows ribosomes to pass into the void volume, so the usual preparation of an S I00 by ultracentrifugation is avoided. The preparation of an S 100 supernatant takes about 3 h, during which time the initiation factor is in solution together with released cellular proteases. It is during this period that most of the IF2T is formed, by proteolytic cleavage after Lys289 [9, 11 ]. The $30 was diluted with buffer H(0) to a final KCI concentration of 100 mM and 50 ml was loaded on a 20 ml column (1.6 x 10 cm) with the anion-exchange matrix Q-Sepharose HP. This sample contains approximately 150 mg crude protein. Ribosomes and unbound proteins were washed through the column in 20 ml of buffer H(100). The adsorbed proteins were fractionated by a 120 ml gradient of 100--400 mM KCI. IF2 was eluted in the range 300--330 mM KCI. Figure 3 shows the chromatogram and an SDS-PAGE analysis of fractions 40--58. As seen these fractions contain IF2tt, IF213 and IF2T as well as EF-Tu. Fractions 45-48 containing the majority of the IF2tx were pooled and the protein content was 8.8 mg, measured as described in Materials and methods. The pool was diluted with buffer H(0) to bring the KCI concentration to 100 mM and loaded onto a 1-ml column of MonoQ equilibrated with buffer H(100). The column was washed with 5 ml buffer H(100) and the adsorbed proteins were fractionated by a 45 ml gradient of 100-300 mM KCI. On this high-resolution material, IF2 was eluted in the range 210-230 mM KCI. Figure 4 shows the chromatogram with the detailed parameters described in the legend. The insert shows an SDS-PAGE analysis of fractions 24-37. The four fractions 29-32 containing essentially all the IF2 were pooled and diluted to 100 mM KCI with buffer H(0).

MonoS Although IF2 is an acidic protein with an isoelectric point of approximately 5.5, an examination of the amino-acid sequence shows that the two N-terminal domains of the molecule have a relatively high content of lysine and arginine residues, resulting in a

Superexpression and purification of E coli IF2 basic local pL This property is used in the choice of the cation-exchange material MonoS for the final step of purification. The dilution pool from the MonoQ chromatography contains 3.2 mg protein. This sample was loaded on a 1 ml column of MonoS equilibrated with buffer H(100). The column was washed with 5 ml buffer H(100) and the adsorbed proteins were fractionated by a 20 ml gradient of 100-250 mM KCI. Pure IF2 eluted as a single peak between 190-200 mM KCI (fig 5). A compilation of the purification results is presented in figure 6 and in table I. In summary, the procedure of cell growth and purification of recombinant IF2 is shown in the flow diagram: Expression:

Azao

fractions

2~.-37 - - -

987 .....

~,'~R

20-

KCt.mM

10-

-300 -200 ,100 ,0

The cells were grown to As.~o= 1.0 at 28°C

$

Induction 20 rain at 42°C

$

Growth continued 2 h at 37°C

$

Cell harvest Opening of cells: $ French pressure cell DNaseI treatment of cell homogenate Purification: $ $30 su~rnatant FPLC: Q-Sepharose High Performance

Fig 4 . Chromatography on MonoQ of the pooled fractions from Q-Sepharose HP in figure 3. Column dimensions: 0.5 x 5 cm = 1 ml MonoQ; sample: 25 ml containing 8.8 mg proteins; flow rate: 1 ml/min; fraction size: l ml. The fractions which were pooled are indicated as the hatched area. Insert: SDS-PAGE analysis of the sample applied to the column (P) and of fractions 24-37.

Azso

P Io---froction's 1&-2/,

MR

0.5i~ ~- 67 K

FPLC: MonoQ

KCI,mM

!i! 4" t3K

$

FPLC: MonoS ~i'~..- 30 K

Characterization and stability of IF2 The identity of the IF2 was confirmed by Western immunoblotting with monospecific rabbit anti-IF2 and by N-terminal amino-acid sequencing (results not shown). Furthermore, the correct C-terminal aminoacid sequence of the recombinant IF2 was determined by carboxypeptidase A and B digestion followed by amino-acid analysis (P Riisgaard, KK Mortensen, HU Sperling-Petersen, unpublished results). In the presence of poly(A,U,G) (0.11 A260 units), GTP (1.5 mM) and IF3 (0.3 l~g), 1.5 l~g of the purified recombinant IF2 shows a ten-fold stimulation of binding of fMet-tRNA~ et (from 0.07 pmol to 0.78 pmol) to 70S ribosomes (15 pmol) which is the generally accepted criterion for full activity in vitro [9]. During isolation of IF2, a smaller form, which we have termed IF2T, appears - presumably by partial proteolysis [9]. The N-terminal amino acid sequence

0.25.

-300 -200 -100

.........

'~o' '~' '~' '~s' 'd '~ . . . . . FRACTION NO,

Fig 5. Chromatography on MonoS of the pooled fractions from MonoQ in figure 4. Column dimensions: 0.5 x 5 cm = 1 ml Mono S; sample: 9 ml containing 3.2 mg proteins; flow rate: 1 ml/ min; fraction size: 1 ml. The fractions which were pooled are indicated as the hatched area. Insert: SDS-PAGE analysis of the sample applied to the column (P) and of fractions 14-24.

988

KK Mortensen et al 1

2

3

t,

In addition, we have discovered another source of degradation which may be of general importance for the purification of proteins. The commercial bovine pancreatic DNase, which is used to degrade the viscous cellular DNA, was found to be contaminated with a protease with trypsin-like properties. Indeed, in an analytical-scale purification experiment where DNase was omitted and cell debris was removed by ultracentrifugation (in a Beckman TL100 benchtop ultracentrifuge), no IF2 T appeared (data not shown).

5

Conclusion

Fig 6. SDS-PAGE analysis of the purification of IF2. Lane 1: crude extract from 500 lxg freshly harvested cells. Lane 2:15 I.tl $30. Lane 3:10 ~1 of the pooled fractions from Q-Sepharose HP. Lane 4:20 l.tl of the pooled fracuons from MonoQ. Lane 5:25 [tl of the pooled fractions from MonoS. of this 65 kDa fragment was determined and compared with that of IF2tx, suggesting that IF2y is generated by proteolytic cleavage of the Lys2sg-Arg290 bond of IF2. Stability tests showed that IF2 is extremely labile in the $30 supernatant whereas, in contrast to what was previously believed, the purified factor is a very stable protein (NR Nyengaard, KK Mortensen, HU Sperling-Petersen, unpublished results). E coli cells carry an outer membrane protease, OmpT [18], with the apparent dibasic amino-acid sequence specificity: Lys-Lys and Lys-Arg. We believe that the presence of OmpT most probably is one major reason for the degradation of IF2 to IF2v in the early stage of purification, immediately after cell lysis. Therefore, it is of highest importance that the factor is separated from membrane debris in a fast initial step.

As shown in table II, the method of superexpression and purification as presented here represents a considerable improvement over methods used hitherto. In this paper we have described in detail the purification of IF2a. However IF213 can be purified by the same procedure. The E coil strain C600[pcls57; pCP40infB] produces the initiation factor IF2 at levels at least 35 times higher than in wild-type E coil strains. Because of the use of FPLC and the new high-productivity chromatographic materials, the purification time has been reduced from 3--4 weeks to less than 8 h and the yield of IF2 is increased 2-3 times.

Acknowledgments The authors thank Dr J Plumbridge, Institut de Biologic Physico-Chimique, Paris, for providing us with the plasmid pBl8-1 and Drs W Fiefs and E Remaut, Laboratorium veer Moleculare Biologie, Riijksuniversiteit-Gent, Belgium for generous gifts of plasmids and strains. We also want to thank Dr M Grunberg-Manago for fruitful discussions and constractire comments to the manuscript. This work was supported by a twinning project grant from the Commission of the European Communities (Contract no SCI*/0194-C(AM)) to HUSP; and FTU-grand from the Danish Natural Sciences Reseea'ch Council (5-17-4.1.04) to HUSP; the Aarhus University Bioregulation Research Centre to KKM and HUSP and the Carlsberg Foundation to KKM; and a postgraduate biotechnology student grant from the Danish Ministry of Education to NRN; and NIH grant GM 40082 to JWBH.

Table I. Summary of purification of IF2 from 1 ! incubation. Step

$30 supematant Q-Sepharose HP MonoQ MonoS

Total protein (mg)

IF2 Purification Yield Time (mg) factor (%) consumption (h)

Table II. Comparison of purification procedures for IF2. Procedure

720 44 16 5

35 15 7.2 5

!

100

1.5

7 9 20

43 21 14

1.5 0.75 0.5

Hershey et al (1977) [101

mg IF2/10 g cells Approximate purity Approximate purfication time

Dondon et al (1985) 1111

This work

0.16 95%

2.0 98%

5.0 > 99%

3 weeks

1 month

8h

Superexpression and purification of E coli IF2

References 1 Remaut E, Tsao H, Fiers W (1983) Improved plasmid vectors with a thermoinducible expression and temperatureregulated runaway replication. Gene 22, 103-113 2 Hershey JWB (1987) Protein synthesis, hi: Escherichia coli and Salmonella typhimurium. Cellular and Molecular Biology (Neidhardt FC, Ingraham JL, Low KB, Magasanik B, Schaechter M, Umbarger HE, eds) American Society for Microbiology, Washington, DC, USA, 613-647 3 Howe JG, Hershey F~B (1982) Immunochemical analysis of molecular forms of protein synthesis initiation factors in crude cell lysates of Escherichia coli. Arch Biochem Biophys 214, 446--451 4 Plumbridge JA, Deville F, Sacerdot C, Petersen HU, Cenatiempo Y, Cozzone A, Grunberg-Manago M, Hershey JWB (1985) Two translational initiation sites in the infB gone are used to express initation factor IF2ot and IF2[~ in Escherichia coll. EMBO J 4, 223-229 5 Potersen HU (1985) Function of tRNA in initiation of prokaryotic translation. Mat Fys Medd Dan Vid Selsk 41, 291-335 6 Plumbridgo JA, Howe JG, Springer M, Touati-Schwartz D, Hershey JWB, Grunberg-Manago M (1982) Cloning and mapping of a gone for translational initiation factor IF2 in Escherichia coli. Proc Natl Acad Sci USA 79, 5033-5037 7 Sacerdot C, Dessen P, Hershey JWB, Plumbridge JA, Grunberg-Manago M (1984) Sequence of the initiation factor IF2 gone: unusual protein features and homologies with elongation factors. Proc Natl Acad Sci USA 81, 7787-7791 8 Sperling-Petersen HU, Mortensen KK (1990) A structural model for initiation factor IF2 from E coli. Protein Eng 3, 343-344 9 Cenatiempo Y, Deville F, Dondon J, Grunberg-Manago M, Sacerdot C, Hershey JWB, Hansen HF, Petersen HU, Clark BFC, Kjeldgaard M, la Cour TFM, Mortensen KK, Nyborg J (198~) The protein synthesis initiation factor 2

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14 15 16 17

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G-domain. Study of a functionally active C-terminal 65kilodalton fragment of IF2 from Escherichia coli. Biochemistry 26, 5070-5076 Hershey JWB, Yanov J, Johnson K, Fakunding JL (1977) Purification and characterization of protein synthesis initiation factors IF1, IF2 and IF3 from Escherichia coli. Arch Biochem Biophys 182, 626-638 Dondon J, Plumbridge JA, Hershey JWB, GmnbergManage M (1985) Overproduction and purification of initiation factor IF2 and pNUSA proteins from a rec~mbinant plasmid bearing strain. Biochimie 67, 643--649 Shazand K, Tucker J, Chiang R, Stansmore K, SperlingPetersen HU, Grunberg-Manago M, Rabinowitz JC, Leighton T (1990) Isolation and molecular genetic characterization of the Bacillus subtilis gone (infB) encoding protein synthesis initiation factor 2. J Bacteriol 172, 2675-3687 Petersen HU, Joseph E, Ullman A, Danchin A (1978) Formylation of initiator tRNA methionine in prokaryotic protein synthesis. In rive polarity in the lactose operon expression. J Bacterioi 135,453-459 Sambrook J, Fritsch EF, Maniatis T (1989) Mole

Superexpression and fast purification of E coli initiation factor IF2.

For the production of large quantities of E coli initiation factor IF2 we have constructed an improved overexpression system. The gene infB was cloned...
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