Genetic engineering of methionyl-tRNA synthetase: in vitro regeneration of an active synthetase by...

Biochirnie 70 (1988) 773-782 (~) Soci~t6 de Chimie biologique/Elsevier, Paris

Research

773

article

Genetic engineering of methionyl-tRNA synthetase: in vitro regeneration of an active synthetase by proteolytic cleavage of a methionyl-tRNA synthetase-/3-galactosidase chimeric protein

P.-Herv6 HIREL, Fran~oise LI~VI~.QUE, Patrice MELLOT, Fr6d6ric DARDEL, Michel PANVERT, Yves MECHULAM and Guy FAYAT*

Laboratoire de Biochimie, Ecole Polytechnique, CNRS UA240, 91128 Palaiseau C6dex, France

(Received 9-11-1987, accepted after revision 13-1-1988)

Summary - - The construction of a family of plasmids carrying derivatives of metG, the gene for E. coli methionyl-tRNA synthetase, is described. These plasmids allow expression of native or truncated forms of the enzyme and easy purification of the products. To facilitate the characterization of modified enzymes with very low catalytic activity, a specialized vector was constructed, in which metG was fused in frame with lacZ, the gene for/3-galactosidase. This plasmid expresses a methionyl-tRNA synthetase- /3-galactosidase chimeric protein, which is shown to carry the activities of both enzymes. This hybrid can be purified in a single step of affinity chromatography for/3-galactosidase. The methionyl-tRNA synthetase moiety can be regenerated by mild proteolysis, thus providing a simple method for purifying and studying mutated proteins.

methionyl-tRNA synthetase / directed mutagenesis / chimeric proteins / expression vector

Introduction

Methionyl-tRNA synthetase is an essential enzyme which catalyzes the specific aminoacylation of both initiator and elongator methionine- specific tRNAs. In Escherichia coil, this enzyme is a homodimer whose 76 kDa subunit is encoded by the metG gene. How the aminoacyl-tRNA synthetases discriminate their cognate tRNAs from the non-cognate remains an important question, since it determines the fidelity of the translation machinery. In order to obtain an understanding of this problem at the atomic level, at least two concomitant approaches are necessary, namely crystallographic studies and genetic engineering of the protein. The methionyl-tRNA synthetase system is particularly suitable for such structure-activity relationship

studies, because crystallographic data-is available for both the enzyme and the tRNA [1], and the metG gene encoding methionyi-tRNA synthetase has been cloned and sequenced [2]. Furthermore, its catalytic mechanism has been resolved [3] and affinity labeling studies have revealed residues close to the binding site for the 3'-aeceptor arm of the tRNA [4]. It is therefore of interest to devise an approach which will allow simple construction of various mutated metG genes and easy purification of the corresponding modified proteins for enzymological studies. This work describes the construction and the characterization of a series of plasmids carrying derivatives of the metG gene designed for this purpose. These plasmids allow production of complete or truncated proteins and exchange of various DNA segments carrying point muta-

*Author to whom correspondence should be addressed.

774 P.-H. Hirel et al.

tions. A metG::lacZ fusion plasmid is also described and the resulting hybrid protein is char- acterized. In vitro proteolyfic cleavage of the chimeric polypeptide is shown to occur at the junction of the two moieties; producing an active methionyl-tRNA synthetase. From this observation, a simple and rapid purification method of mutant proteins can be derived.

Materials and methods

General techniques General molecular biology .techniques were as described in Maniatis et al. [5]. Bacterial strains and plasmids are listed in Table I. Restriction endo- nucleases and DNA modifying enzymes were obtained from Boehringer Mannheim, Pharmacia, BRL or Appligene (Strasbourg, France). Incubations were performed as described by the supplier. The DNA sequence was determined by the dideoxy chain terminator method [6, 7]. DNA fragments were purified by size exclusion chromatography [8].

Derivation of pNA V plasmids Plasmids of the pNAV family are pUC12 derivatives [9]. pUC12 polylinker was modified as follows: firstly, an Xhol linker (5'-CCTCGAGG-3', Pharma- cia) was inserted into the filled-in HindlIl site. Secondly, a universal translation terminator cartridge (5'-GCTI'AATI'AATFAAGC-3', Pharma- cia) was ligated into the Hindll site. Thirdly, the Pstl site was deleted by treatment of the PstI 3'-protrud- ing termini with T4 DNA polymerase and ligation. The resulting plasmid was called pNAV3 (Fig. 1). The modified polylinker structure was checked by DNA sequencing.

Plasmid pNAV4 was obtained by insertion of the 4.8 kb EcoRI-BamHl fragment of plasmid pX1 [2] between the corresponding sites of pNAV3 (Fig. 2). This plasmid carries most of the metG gene and it~ upstream region. Phage M13mpl9x1 was constructed by ligating a Scal-HindllI fragment carrying metG 5' terminus in M13mpl9 cleaved by HindlI and HindIll. The BamHI site of the polylinker was then eliminated by filling-in with the Klenow fragment of E. coli DNA polymerase and religation. A BamHl -Alul fragment carrying the Y-end of metG was clon-

Table !. Bacterial strains and plasmids.

Strain or plasmid Relevant genotype or scorable markers Reference

Strains

JM101 supE thi-1 A(lac-pro) [7] F' (traD36 iaclq proA B lacZAM 15)

J M l f l l T r ¢ . . . . . l l ~ A 1 / q t l - - - A e L _ _ l ~ d a r l . . . . . . . . . . . . . . . . . . . O o . l i l ~ , e (]A3 J I V g J [ W J I ~ J" ~'L.-,~'I.. ,1U, , . ~ f t J l ] l L g ~ , . I I ] / ' ! . . / l l~!

IBPCI 11 F- A(lac-pro) gyrA rpoB metB argEam [20] ara supE recA 1

Plasmids

pX1 metG, TetA, carries a 14.5 kb chromosomal [2] insert in the Pst! site of pBR322

pUC12 bta 19] pMC 1871 'lacZ, bla, carries a/3-galactosidase [ 10]

gene cartridge between multiple restriction sites

bla, derivative ofpUC12

metG', bla

metG', bla

metG, bla

metG::lacZ, bla

pNAV3

pNAV4

pNAV6

pNAV7

pNAV9

this study

this study

this study

this study

.this study

Methionyl-tRNA synthetase expression vectors 775

Acc AA ~. TCC AGC TCC CCC GGG CAT CCT CTA GAG TCG CTT ,EAT TAA TTA AGC G]~C CCC CCA ACC TCC TCG /~CG AGC 2"

t t t t t I I r I Xhol linker EcoRI Sact Sinai BarnHI Xbal universal translalion in Hin¢ltl site

terminator (in Hindll site)

I BamHI S m a ~ Pstl Site T4 +polymerase deleted

E c o R ~ ~ / / Xhol linker I ~te

p U C 1 2 repl icon

Fig. 1. Structure of pNAV3, a pUC12 derivative with a modified polylinker containing a universal translation terminator.

ed into Ml3mp9, between the BamHI and Sinai sites of the polylinker. An XhoI linker was then inserted into the filled-in EcoRI site to yield M13mp9X1BA2. This reconstructed two EcoRI sites, one on each side of the XhoI linker.

Advantage was taken of the EcoRl site of Ml~mpltlx1 n n l v l l n k o r t n r p n l ~ eh~ K ~ I D I - - / . J ' ; - - A l l l

fragment of pNAV4 by the corresponding one of M13mpl9xl. The resulting plasmid, pNAV6 carries only a short DNA segment upstream from metG (67 bp) (Fig. 3). To reconstruct an intact metG gene, the BamHI-Xhol fragment of M13mp9X1BA2 was ligated between the BamHl and Xhol sites of pNAV6. The resulting plasmid was called pNAV7 (Fig. 4). Using the lacZ gene cartridge carried by plasmid pMC1871 [10], a plasmid carrying an in frame metG::lacZ protein fusion, pNAV9, was constructed by insertion of the purified BamH1 fragment of pMCI871 carrying lacZ into the unique BamHl site of pNAV6, internal to metG (Fig. 5). Colonies of strains carrying pNAV9 are deep blue on 5-bromo- 4-chloro-3-indolyl-13-D-galactopyranoside indicator plates.

Purification of overproduced methionyl-tRNA syn. thetase Strain JMI01Tr was used to host the methionyl- tRNA synthetase (MTS) expression vector pNAV7. The cultures were carried out in a Biolafitte fermen- tation unit. 15 1 of M63 minimal medium [11] supple- mented with 15 p.M ZnSO4 (to insure a full saturation of the high affinity zinc site of methionyl-tRNA syn-

thetase [12]) were inoculated with cells at a final optical density of 0.1 A650 units. At 0.2 A650 units, IPTG (iso- propyl /3-D-thiogalactopyranoside) was added at a final concentration of 1 mM. Cells were harvested in the exponential phase (1.5 Ars0 units). 100 g (wet weight) of bacteria were resuspended in 3 1 of buffer A (20 mM Tris-HCi; i0 mM 2-mercaptoethanol; pH 7.6) and subjected to ultrasonic disintegration. After streptomycin precipitation (3% + w / v ) , ammonium sulfate fractionation (50% of saturation), and dialysis of the pellet against 10 1 of 20 mM potassium phosphate buffer, 10 mM 2-mercaptoethanol, pH 8.0, the extract was fractionated by molecular sieving on Sephacryl S-300 (4.4 × 90 cm, 40 ml-h-~). The eluate was loaded onto a DEAE-tr isacryl M column (4.4 x 20 cm, 66 ml.h-l), and a linear salt gradient was then applied (20-80 mM KCI, 2 x 3 !). The pooled fractions were dialyzed against 20 mM potassium phosphate buffer (pH 6.8) and loaded onto a Biogel HTP column. MTS was eluted by application of a linear phosphate gradient (20-200 mM potassium phosphate, pH 6.8, 2 x 1.5 I, 100 ml-h-J). The pure enzyme was stored at -20oC, after dialysis against buffer A containing 55% ( v / v ) glycerol.

Assays of tRNA aminoacylation and A TP -PP i exchange activities were performed as described pre- viously [13]. Pure enzyme concentrations were derived from the absorbance measurement at 280 rim, using the following specific extinction coefficients: 1.39, 1.72 and 1.78 OD cm2-mg -I for native MTS, the tryptic fragment of MTS (MTSt), and the MTS-/~-galactosidase hybrid, respectively.


1 metG gene

z.,.\\\',,~ pBR322 DNA EcoRI Pstl Hindlll Pstl BamHI

_[~ I I I

namHI Pstl S~te T4 -polymerase delelcd

EcoR, ~. ~ : ~ : ~ , ~ / , Xhol linker f / . .,o=,.-.

univers~.i translalion terminator

replicon

ndlll

c0,, .%.

Fig. 2. Construction of pNAV4, a plasmid expressing an active truncated methionyl-tRNA synthetase. The 4.8 kb EcoRI-BamHl fragment of pX1 [2], carrying the 5' terminal of metG, was inserted between the corresponding ~ites of pNAV3.

Purification of the MTS-13-galactosidase hybrid protein [BPC111 cells containing the pNAV9 plasmid were grown at 37oC in 2 1 of LB medium containing 50/zg of ampicillin / ml. Cells were harvested at the stationary phase. Bacteria were resuspended in 200 ml of standard buffer (10 mM Tris-hydrochloride; 10 mM magnesium chloride; 1 mM dithioerythritol (DTE); 0.1 mM ethylenediaminetetraacetic acid (EDTA); pH 7.6) and disrupted by ultrasonic disintegration. Nucleic acids were removed by streptomycin precipi tation (3%, w / v ) . Proteins were precipitated from the supernatant at an ammonium sulfate concentra tion of 40% of saturation. The pellets were redissolv

ed in 15 ml of standard buffer (alternatively, these last 3 steps could be omitted without affecting the overall efficiency of the purification procedure). NaCi was then added to a final concentration of 1.6 M. One-step purification of the ~-galactosidase fusion protein was performed on a 2 ml column prepared by coupling p-aminophenyl-/3-a-thiogalactopyranoside to CH-Sepha rose 4B as described by Ullman [14]. After loading and washing, the hybrid was eluted with 0.1 M sodium borate (pH 10.3), the pooled fractions were either neutralized with 0.5 M

Pstl Scal Hindlll Pstl BamHI ":.cl

I I I I I I

Clone the ScaI-Hindlll fragment between the Hindll and Hindlll sites of M13mpt9

I Clone tile EcoRI-Hindlll fragment between the corresponding sites of pNAV4

EcoRI ~ Hindlll

Xhol

Fig. 3. Derivation of pNAV6. This plasmid is similar to pNAV4, except that the chromosomal region upstream from metG is only 67 bp long, thus placing this gene under the direct control of the lac promoter. It was constructed by a two-step procedure: first an ScaI-HindIlI fragment containing the 5' part of metG was cloned between the Hindll and Hindlll sites of Ml3mpl9, then we took advantage of the EcoRl site of the M13 polylinker upstream from the insert to replace the [aNAV4 EcoRI-HindlII fragment by this shorter fragment.


Pstl

I Seal

L Hindl l l Psll BamHI Accl

Cut by Alul. digest the 3" pmlmding end, BamHI oat. ~se~t ~ tw~n the Sinai BateHI sit~ of M13mp9; insert a Xhol linker in tho l~lBd-in EcoR~ she.

/ e,"E0o.,

I Clon¢l the BamHI-Xhol fragment between the ¢otreSpoczding sites of pNAV6

EcoRI Hindlll ~ BamHI

Fig. 4. Construction of pNAV7, an MTS overproducing plasmid. This plasmid was constructed by inserting the 3'- terminal part ofmetG after the BamHl site of pNAV6, thus reconstructing an intact gene.

phosphate buffer (pH 6.5) or precipitated with ammonium sulfate (70% of saturation). The hybrid protein was stored at -20oC, after dialysis against standard buffer containing 55% (v/v) glycerol. B- Galactosidase activity was assayed according to Mil- ler [15].

R e s u l t s

It is well-documented that upon mild treatment with trypsin, native dimeric MTS (2 × 76 kDa) is transformed into a fully active monomeric form, MTSt, of M r 64 000, which retains the

same N-terminus as the native enzyme [16]. It is of interest to determine precisely the minimal regions essential for either activation of methionine or t R N A aminoacylation activities. A direct approach to this probk, m is to produce and study polypeptides of various lengths truncated on the C-terminal side. In addition, the only crystallographic structure available is that of MTSt, the active tryptic fragment of MTS [1, 17], and not that of native MTS. It is therefore interesting to produce genetically engineered analogs of MTSt, both for purification and analytical pur- poses. In a first step, the length of the shortest molecules retaining methionine activation a n d / o r t R N A aminoacylation activities can be determined following progressive BAL31 dele- tion of the 3' end of the metG gene.

EcoRI Hindlll

Xhal

J Insert a lacZ BamHI carlddge in the Bamlql sile of pNAV6

EcoRI ~ Hindlll

~ ~ ~ ~ BarnHI

Xhof

Fig. 5. Derivation of pNAV9. Insertion of the lacZ cartridge of pMC1871 [10] into the BamHI site of pNAV6 yielded an in frame metG::lacZ protein fusion.


Designing a m e t G vector for production o f truncated methionyl-tRNA synthetases

To facilitate the construction of several scores of truncated genes, a specialized shuttle vector was designed. Such a vector was to contain metG up to its BamHI site, and all the useful sites for enzymes which cut singly within the metG gene (HindllI and PstI) had to be removed from this plasmid. It was derived from pUC12. The polylinker of this vector was modified to eliminate the undesired sites. In addition, an XhoI site (not present within metG) was added in place of HindllI. Since 3'-truncated fragments of metG were to be cloned into this vector, a universal translation terminator was also inserted into the polylinker immediately downstream from the BamHI site, in order to prevent read-through translation into plasmid sequences. This vector, called pNAV3, is shown in Fig. 1.

A 4.276 kb fragment from plasmid pX1 [2] carrying the first 621 codons of metG was inserted between the EcoRI and BamHl sites of pNAV3. The resulting plasmid, pNAV4 also carries the metG promoter, 1.71 kb of chromosomal DNA upstream from metG and an EcoRI- PstI fragment originating from pBR322. Therefore, we expected that the truncated metG gene carried by pNAV4, which has a coding capacity of 70 kDa, larger than MTSt, would express an active polypeptide. Both aminoacylation and A T P - P P i exchange activities of methionyl-tRNA synthetase where measured in JM101 cells carrying pNAV4. A small but significant increase in both activities was observed (1.6 times). This factor was not modified when the lac promoter carried by pNAV4 was induced with IPTG. The fact that the increase in MTS activity was rather limited could be due to a reduced activity and / or stability of the trunc-

ated polypeptide a n d / o r interference of the DNA region upstream from the gene with its expression. The truncated MTS expressed by pNAV4 is monomeric (data not shown). Fur- thermore, pNAV4 is able to complement the methionine auxotrophy of a metG mutant. Therefore, it can be concluded that the monomeric truncated form of MTS is able to sustain tRNA Met aminoacylation in vivo.

In order to obtain a higher level of expression, the DNA fragment carrying the 5' part of the metG gene and the upstream regions was replac- ed by a smaller one (1930 bp instead of 4276 bp). In this plasmid, pNAV6, the chromosomal region upstream from metG is reduced to 67 bp, thus placing the gene under the direct control of the lac promoter. MTS activity was measured in IPTG-induced and uninduced JM101 cells carrying pNAV6 and compared to JM101 /pUC12. Cells carrying pNAV6 overproduce MTS activity 3.5 times and upon IPTG induction, this factor reaches 5.9. As shown in Fig. 3, the pNAV6 plasmid contains a set of single sites which allow easy replacement of any region of the metG gene. Moreover, a complete gene can be reconstituted by inserting a BamHI-XhoI fragment encoding the C-terminal part of the enzyme.

Construction of an expression vector for native methionyl-tRNA synthetase overproduction

An expression vector carrying the complete metG gene was constructed by inserting a DNA fragment carrying the 3' terminal part of metG between the BamHl and Xhol sites of pNAV6. The resulting plasmid, pNAV7, carries a 2395 bp insert encompassing the whole metG gene. JM101Tr cells carrying pNAV7 overproduce

l'able II. Purification of overproduced native methionyl-tRNA synthetase.

Total protein Total activity Yield (mg) (units × 10 -6) (%)

Specific activity (units / mg)

Crude extract 6 000 24 100 4 000 (NH4)2SO4 precipitate 2 257 22.3 93 9 880 Sephacryl S-300 eluate 1 122 20 89 17 825 DEAE-trisacryl M eluate 688 18.6 79 27 034 Biogel HTP eluate 275 18 75 65 454

The,enzyme activity was followed using the tRNAMct aminoacylation activity. One unit corresponds to the enzyme activity that catalyzes tile transfer of 1 pmol of [14C]methionine onto tRNA/s at 25oC in a final reaction volume of 100/zl.


native MTS 20-fold, and upon IPTG induction this factor reaches 80-100-fold. Crude extracts of cells carrying this plasmid subjected to SDS-polyacrylamide gel electrophoresis (PAGE) exhibit a major overproduced polypeptide band corresponding to the M r of MTS. This overproducing strain has been used for large scale production of MTS from 15 1 fermentor cultures. From 100 g of IPTG-induced bacteria (wet weight), 270 mg of homogeneous native dimeric enzyme were obtained with a final yield of 75%, following the procedure described under Materials and Methods (Table II). A frac- tion of the purified native enzyme (100 mg) was used to produce the active monomeric form of MTS by mild tryptic proteolysis. After separa- tion of the digestion products by hydroxyapatite chromatography, 50 mg of pure MTSt were obtained, which were shown to crystallize in a form suitable for X-ray analysis.

Finally, the metG containing insert can be easily excised by an EcoRI digestion and trans- ferred into any other vector more convenient for oligonuc!eotide-directed mutagenesis, such as M13-derived vectors (M13mp's or Bluescript).

Construction of a vector expressing a hybrid M T S - B-galactosidase

It is often of particular interest to characterize genetically-engineered enzyme forms exhibiting a very low catalytic activity. However, the purification of such enzymes is tricky, since it is necessary to obtain these mutant proteins free from wild type chromosomal product contami- nation. To overcome this difficulty, one possibi- lity is to express the mutant enzyme as a hybrid protein containing an active /3-galactosidase

moiety. Such a chimeric protein can be easily purified in a single step by affinity chromatography specific for the/3-galactosidase moiety.

As described above, it has been shown that a monomeric form of methionyl-tRNA synthetase composed of less than 600 amino acids completely retains both the activation of methionine and tRNA aminoacylation activities. Therefore, the first 621 codons of metG were fused in frame to the last 1017 codons of /3-galactosidase. As shown in Fig. 5, pNAV9 was constructed by inserting a lacZ cartridge into the BarnHl site of pNAV6.

Overproduction and purification of the hybrid protein

The fusion polypeptide expressed by pNAV9 (Mr 185 500) could be detected by S D S - P A G E of crude extracts of pNAV9 carrying cells. To assay MTS and /3-galactosidase activities, pNAV9 was transformed into IBPC111, a Alac, recA strain. Indeed, an overproduction of MTS aminoacylation activity was observed, ranging from 2 times in the exponential phase to 10 times in the stationary phase. This overproduction factor paralleled the/3-galactosidase activity mea- surements. When the results were expressed in terms of protein concentration in the extract, using the known specific activities of either pure MTS or /3-galactosidase, compatible figures were obtained (Table !!!).

In order to characterize more fully the chimeric polypeptide encoded by pNAV9, a medium scale purification was undertaken. The tech- nique used requires a single chromatographic step on a 2 ml column of p-aminophenyl-/3-o- thiogalactopyranosidyi-Sepharose (Fig. 6), an affinity substrate of /3-galactosidase [14, 18].

Table !!i. Concentration of MTS-fl-galactosidase hybrid in E. coli extracts carrying pNAV9.

/3-Galactosidase Aminoacylation ATP- PPi exchange (nM) (nM) (nM)

Small scale culture 20 24 33 (exponential phase)

Fermentor culture 173 105 n.d.

Crude extracts were prepared by ultrasonic disintegration of the equivalent of 40D6s0 of cultured cells suspended in 1 ml of 20 mM Tris-NCl, pH 7.6; 0.1 raM EDTA; 0.1 mM//-mercaptoethanol. Enzymatic assays were performed as described under Mate- rials and Methods, and the resulting activities were converted into enzyme concentrations, using the known specific activities of MTS and B-galactosidase, and assuming they were conserved in the hybrid protein. (n.d.: not determined).


3 '

1

o

800 F 20,000 ----~

- 600 1 5 , 0 0 0

-400 I 10,000

[ -2oo ~, [ ~,ooo

. . . . . . . 0 LO 3O 10 20 1'40 50

Vo;um~ -=- ,~8o 123 "8- Beta-galactosidase -Q- Aminoacylation

Fig, 6. Single step purification of the MTS-/3-galactosidase hybrid. Shown is the elution profile of the 2 ml p-aminophenyl-/3-D- thiogalactopyranosidyl-Sepharose column. A pH step (20 mM Tris-HCI, 10 mM MgCI2, 1 mM DTE, 0.1 mM EDTA, 1.6 M NaCI, pH 7.5 --* 0. l M sodium borate, pH 10.3) was applied after washing with 20 column vol. (arrow). As can be seen on the figure, the/3-galactosidase activity co-elutes with the MTS aminoacylation activity. The resulting hybrid is >90% pure, as can be seen on Fig. 7B, lane 2.

Upon elution from the column, the/3-galactosidase activity decreases rapidly, and is completely lost after concentration and dialysis. The MTS aminoacylation activity is, however, fully retained by the hybrid protein. Approximately 7 mg of MTS-B-galactosidase were thus obtained from a 2 1 culture, with an estimated yield of 80%. The purity of the chimeric protein was con- troled by S D S - P A G E . The MTS kinetic para- meters of the hybrid where determined and compared to those of the tryptic fragment of MTS (Table IV). The MTS-~-galactosidase hybrid is

Table IV. Aminoacylation and methionine activation initial velocities of MTS-/3-galactosidase hybrid and MTSi.

Hybrid MTSt (s l) (s ')

Activation 30.0 60.1) Aminoacylation 0.8 4

The amount of hybrid used in the test was deduced from its mass extinction coefficient (~-~) which was approximated as the weighted average of MTSt and/3-galactosidase extinction coefficients.

one half as active as far as methionine activation is concerned and one fifth as active in aminoacylation. This difference could be due to a steric hindrance of the/3-galactosidase moiety, inter- fering with the access of tRNA to its site.

An MTSt molecule can be regenerated from the hybrid protein

Since the MTS part of the hybrid is a little longer (621 residues) than the tryptie fragment of native MTS [14], and since the hybrid carried MTS activity, thus indicating that it was correctly fold- ed, it was of interest to determine whether MTS could be released from the chimeric protein by proteolytic cleavage. For this purpose, a kinetic study of the trypsinolysis was performed. As shown in Figure 7B, the hybrid was very quickly hydrolyzed into a polypeptide having the same molecular ratio as/3-galactosidase and two smaller bands which were also observed in the course of MTS trypsinolysis. The larger one corresponds to an intermediate species, and the smaller one is MTSt. Upon longer incubation times, the /3-galactosidase polypeptide disappeared, whereas the intermediate species was converted into a protein comigrating with MTSt. These results confirmed the above predictions and showed that cleavages occurred at similar posi-


tions in both native MTS and the hybrid protein. In addition, the fact that the fl-galactosidase polypeptide was completely digested during the course of the proteolysis was consistent with the loss of the corresponding catalytic activity. This probably reflects a denaturation caused by the elution procedure at basic pH.

s-1

A

During proteolysis, both activities of MT5 were monitored (Fig. 7A), Interestingly, cleavage of the hybrid resulted in a reactivation (1.5 x) of aminoacylation capacity. This observation is consistent with the assumption that the smaller species yielded by trypsinolysis corresponds to the tryptic fragment of MTS and

12

o

1.0

i o.[ 0.8 - •

u 1 0 0 , , 2 0 0

40

°

.¢.

O 1 0 0 2 0 0

B ~ 2 3 4 s s 7 8

Hybrid

sidase

. . ..

<1.-. MTSt

Fig, 7. Regeneration of MTSt by mild proteolysis of the MTS-/~-galactosidase hybrid. A0 Both MTS activities were monitored during the course of hybricl trypsinolysis. The initial aminoacylation and ATP-PP~ exchange velocities are shown as a function of the incubation time for a 1 / 1000 (w / w) trypsin / hybrid ratio. Maximal reactivation was observed after an ~20 rain incubation time. B. $DS-PAGE analysis of the proteolysis kinetics. Lane l • native/3-galactosidase; lane 2: purified MTS-/~-galactosidase hybrid; lanes 3 -7 proteolytic products observed after 15, 30, 60, 90 and 120 rain of incubation with trypsin (1 / 1000 w/w); lane 8: purified MTSt.


agrees well with the above hypothesis of steric hindrance of the tRNA site by the /3-galactosidase moiety in the hybrid.

Conclusion

This paper reports the construction of specialized expression vectors allowing overproduction and isolation of native and genetically engineered methionyl-tRNA synthetase. A plasmid encoding a truncated polypeptide, similar to the crystallized tryptic fragment of methionyl-tRNA synthetase, has been constructed. This plasmid is particularly suitable for site-directed mutagenesis, since the mutant monomeric enzyme it will express will be easily separated from the non-dispensable dimeric MTS encoded by the chromosome. An important observation arises from the fact that this plasmid is able to express in vivo an active monomerie form of MTS. It was known that the C-terminal part of MTS could be removed by in vitro proteolysis of the native dimeric enzyme without affecting the catalytic activity. This study now demonstrates that the C- terminal part of the polypeptide is also dispensable for a proper in vivo folding of the 550 N- terminal amino acids. Therefore, it can be concluded that the N-terminal domain is able to fold by itself, i.e., independently of the C-terminal domain.

A vector expressing a hybrid MTS-/3-galactosidase protein has been constructed. The fusion protein retains both MTS and B-galactosidase activities. It can be purified to homogeneity in a single chromatographic step. Interestingly, upon mild trypsinolysis, an MTS fragment indistinguish- able from the crystallized MTSt can be regenerated from the hybrid protein, while the/3-galactosidase moiety is rapidly and completely degrad- ed. During the course of the proteolysis, both activation and aminoacylation specific activities increased to reach maximal values close to those of pure MTSt. The final digestion pattern of the hybrid protein is very similar to that of the native dimeric MTS. This further supports the idea that the N-terminal sequence (550 amino acids) contains all the information required for the proper folding of the active part of the enzyme. To conclude, this fusion vector system provides a unique tool to achieve purification and biochem- mical characterization of genetically engineered methionyl-tRNA synthetases devoid of enzymatic activity.

Acknowledgments

The authors wish to thank Pr. Sylvain Blanquet in whose Laboratory this work was carried out for con- stant interest and support. Dr. Patrick Lemaire is acknowledged for his help at the initial stage of this study. This work was supported by grants UA240 and ATP 6931 from the CNRS. PHH was a recipient of a studentship from Rhone-Poulenc Recherches.

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Genetic engineering of methionyl-tRNA synthetase: in vitro regeneration of an active synthetase by...

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