Protein farnesyltransferase: production in Escherichia coli and imrmmoaffinity purification of the heterodimer from Saccharomyces cerevisiae

Gene, 132 ( 1993) 4 l-47 Elsevier Science Publishers

41

B.V.

GENE 07299

Protein farnesyltransferase: production in Escherichia coli and immunoaffinity purification of the heterodimer from Saccharomyces cerevisiae (Farnesyl diphosphate; prenylation; yeast; H,,, protein; Michaelis constants)

Matthias P. Mayer*, Glenn D. Prestwich*, Julia M. Dolence, Pamela D. Bond, Hong-yu Wu and C. Dale Poulter Department of Chemistry. University of Utah, Salt Lake City, UT 84112, VISA Received by G.P. Livi: 26 January

1993; Revised/Accepted:

1 April/8 April 1993; Received at publishers:

I June 1993

SUMMARY

Protein farnesylation in Saccharomyces cerevisiae is mediated by a heterodimeric enzyme, protein farnesyltransferase (PFTase), encoded by the genes RAM1 and RAMZ. A series of plasmids for the expression of RAM1 and RAM2 in Escherichia coli was prepared and evaluated. Maximal production of functional PFTase was seen in strains containing a multicopy plasmid with a synthetic operon in which the RAM1 and RAM2 structural genes were translationally coupled by overlapping TAATG stop-start codons and by locating a ribosome-binding site near the 3’ end of the upstream gene. This was accomplished by an insertional mutation at the 3’-end of RAM1 that embedded an AGGAGGAG sequence within codons for the tetrapeptide, QEEF, added to the end of the Ram1 protein. The QEEF C-terminal motif in the Ram1 subunit of PFTase facilitated purification of the enzyme by immunoaffinity chromatography on an anti-cl-tubulin column prepared using monoclonal antibodies that recognized a tripeptide EEF epitope. Heterodimeric recombinant yeast PFTase::QEEF (re-PFTase::QEEF) constituted approximately 4% of total soluble protein in induced cells and was readily purified 25-fold in two steps by ion exchange and immunoaffinity chromatography in an overall 25% yield. Michaelis constants for farnesyl diphosphate (FPP) and H,,, protein (modified to contain a yeast a-mating factor PACVIA sequence at the C terminus) were 5.5 and 15 PM, respectively; the k,,, was 0.7 s-l.

INTRODUCTION

Protein prenyltransferases catalyze alkylation of Cys residues near the C terminus of a variety of polypeptides, including a number of proteins involved in signal transCorrespondence to: Dr. CD. Poulter, Department of Chemistry, University of Utah, Salt Lake City, UT 84112, USA. Tel. (I-801) 5816685; Fax (I-801) 581-4391; e-mail: [email protected] *Present addresses: (M.P.M.) University of Geneva, Department of Medical Biochemistry, Centre Medicale Universitaire, 121 I Geneva 4, Switzerland. Tel. (41-22) 702-5514; e-mail: [email protected]; (G.D.P.) Department of Chemistry. State University of New York, Stony Brook, NY 11794, USA. Tel. (l-516) 632-7926; e-mail: Glen.Prestwich(alunysb.edu Abbreviations:

aa. amino

acid(s):

Ap, ampicillin;

BME,

2-mercapto-

duction and intracellular transport (Clarke, 1992). At least three different enzymes catalyze prenylation of polypeptides (Moores et al., 1991). Protein farnesyltransferase (PFTase) selectively adds a farnesyl residue to the Cys in a tetrapeptide CAAX C-terminal motif, where A is an ethanol; bp, base pair(s); cGMP, cyclic guanosine monophosphate; E.. Escherichia; FPP, farnesyl diphosphate; GGPP, geranylgeranyl diphosphate; IPTG, isopropyl-p-D-thiogalactopyranoside; kb, kilobase or 1000 bp; LB, Luria-Bertani (broth); nt, nucleotide(s); ORF. open reading frame; PACVIA, Pro-Ala-Cys-Val-Be-Ala; PAGE, polyacrylamide-gel electrophoresis; PCR, polymerase chain reaction; PFTase, protein farnesyltransferase; PGGTase, protein geranylgeranyltransferase; PMSF, phenylmethylsulfonyl fluoride; RBS, ribosome-binding site(s); re-, recombinant; S., Saccharomyces; SB, super broth; SDS, sodium dodecyl sulfate; ss, single strand(ed); wt, wild type; [I. denotes plasmid-carrier state; ::, novel junction (fusion or insertion).

42 aliphatic

aa and X is A (Ala), M, Q, or S. Protein

nylgeranyltransferase alkylation sequences,

I (PGGTaseI)

catalyzes

of Cys by a geranylgeranyl group in CAAX where X is L, N, and sometimes F. A third

enzyme, PGGTaseII, is responsible lation of CC and CXC C-terminal were recently

purified

approx.

brain tissue by affinity chromatography ing immobilized

peptides

sequences.

Subsequently,

3000-fold

purification

(Moores

containing

et al., 1991)

60000-fold

from

on columns

bear-

C-terminal

CAAX

Gomez et al. (1993) reported of

Saccharomyces

pHYW3 taining

directed

synthesis

the CVIA

factor (Clarke, The recombinant

protein

of yeast

con-

a-mating

was purified

to >90%

homo-

the

cerevisiae

for yeast PFTase. (b) Construction and evaluation of expression plasmids for

RAM1 and RAM2 Plasmids constructed

for the synthesis from

pMPMS-1

RAMZ, the genes that encode

tives of pARC306N.

for CI and B

subunits of the enzyme. Our aim was to construct a system for producing larger quantities of yeast PFTase for kinetic and structural studies. Although RAMI and RAM2 have been expressed in E. coli, the activity of heterodimeric PFTase was low (He et al., 1991). Initial attempts by us to overproduce the enzyme in E. coli were hampered by the tendency of the Ram1 and Ram2 proteins to form inclusion bodies when the genes were expressed individually or coexpressed in unequal amounts. We now report the construction of multicopy plasmids containing a synthetic operon that couples expression of RAM1 and RAM2 from a single, inducible promoter. In addition, a C-terminal EEF a-tubulin epitope was fused to the Ram 1 protein to facilitate purification by immunoaffinity chromatography. Overproduced recombinant mutant yeast PFTase constitutes approximately 4% of total cellular protein in our overproducing strains.

RESULTS

H,,, protein

motif

1992) in place of its wt CAAX sequence.

PFTase to near homogeneity from a yeast transformant harboring multicopy plasmids containing RAM1 and the proteins

of mutant

C-terminal

geneity from fresh cells according to the procedure of Manne et al. (1985). This material was used as a substrate

for the geranylgeranysequences.

Rat (Reiss et al., 1990) and bovine PFTases

gera-

a related

AND DISCUSSION

(a) Construction of an expression plasmid for H,,,-PACVIA and purification of the protein The wt gene encoding human H,,, protein (Der et al., 1982) was modified by PCR using Taq DNA polymerase and the appropriate primers to introduce an NdeI restriction site encompassing the ATG start codon, to replace the CAAX box at the C terminus of the protein with the hexapeptide PACVIA derived from the C terminus of unmodified yeast a-mating factor (Clarke, 1992), and to introduce a PstI restriction site downstream from the stop codon. The modified ORF was cloned between the NdeI and PstI sites of pTTQl8N, a derivative of pTTQl8 (Pharmacia, Alameda, CA) with a unique NdeI site located 4 bp downstream from the RBS. The insert in the resulting plasmid, pHYW3, was sequenced (Sanger et al., 1977) to verify its structure. Upon induction with IPTG,

the

and pMPM5-2

of PFTase

RAM1

in E. coli were

RAM2

and

(see footnote

of the entries in Table I, except pGPl14-2/l/2, This expression

ORFs

in

a in Table I). All are deriva-

vector contains

the

strong rec7 promoter, which is a hybrid of the E. co/i recA promoter and the phage T7 gene 10 leader sequence (Olins et al., 1988). The gene 10 leader contains an RBS and a start codon (ATG) that forms part of an NdeI restriction site. As discussed below, most of the Ram1 and Ram2 proteins formed inclusion bodies in E. co/i strains transformed with pARC306N derivatives pMPM7-1 or pMPMl6-2 containing the ORFs for RAM1 or RAMZ, respectively. Attempts to obtain active PFTase by combining the small amounts of cytosolic Ram1 and Ram2 from transformants were unsuccessful. We then decided to construct synthetic operons containing RAM1 and RAM2 and were drawn to studies of the trp operon in E. coli. When derepressed, the operon directs synthesis of approximately equal amounts of five different proteins from a single mRNA. The five structural genes - trpE, trpD, trpC, trpB, trpA - are translationally coupled (Yanofsky and Crawford, 1987). Each ORF is preceded by AGGAG, AGGA, or AGGG RBS sequences separated from the translation initiation codon by a 7to 9-bp A + T-rich sequence. The RBS are located in noncoding spacers or within the preceding structural genes. In two fusions, trpE/trpD and trpB/trpA, the last bp of the stop codon overlaps the first bp of the start codon. These structures give tightly coupled translation of both structural genes. Plasmids pMPM7-1 and pMPM16-2 were used to construct a series of synthetic operons containing RAM1 and RAM2 ORFs in a pARC306N background. The nt sequences at the junctions between ORFs shown in Table II were constructed by PCR. In pMPM9-1,2, RAMI and RAM2 were separated by a 20-nt spacer that contained an RBS (AGGAG) 5 bp upstream from the start codon for RAMZ. In pMPMll-l/2 (RAMlIRAM2) and pMPM13-2/l (RAM2/RAMl), the stop codon of the upstream ORF overlapped the start codon of the downstream ORF by a single bp. Plasmid pMPM14-2/l/2 contained a copy of RAMI flanked by copies of RAM2 with

43 TABLE

I

Plasmids

synthesis for production

Plasmid”

ORFI

into pARC306N

_ _

_

RAM2 RAM2

_

site 3 bp downstream

_ _

ment (SeaKern, FMC, Rockland, ME, USA) was cloned between the EcoRI and XhoI sites of pBluescript-SK(+) (Stratagene) to give

pMPMII-l/2 pMPMl3-2/l

RAM1 RAM2

pMPM14-2/l/2 pGP14-2/l/2

RAM2

RAMI RAMI

RAM2

pMPMS-I.

RAM2

RAM2

pGPl14-2/l/2

RAM2

RAMI

RAM2

published mutation

RAM1

“E. coli strain DH5a (recA_, F-, gyrA96, thi-I. hsdR17[r;, supE44, relA1) was used for cloning. Strains DH5cl and JMlOl

ml], (A[lac-

pro], thi, strA, endA, sbcBl5, hsdR, subElF’, traD36, proB+. lacZAM IS) were used for expression. E. coli strains CJ236 (ung-, were purchased

pT7-DPRl

was provided

from Bio-Rad

mutagenesis

by J. Rine. Plasmid

pBH57

laclq, dut -)

(Richmond,

experiments.

S. Powers. Methods: Typical PCR reactions contained buffer/O.2 mM dNTPs/I pg of each primer DNA/250

Plasmid

was a gift from Taq polymerase ng of template

DNA/5 units of Taq DNA polymerase (Stratagene, La Jolla, CA, USA) in a total volume of 100 11. The mixture was overlaid with 50 ptl of mineral oil and incubated in a Coy Tempcycler with the following program: included

5 s at 95.5”C, 5 s at 37”C, 3 min at 77°C. The first cycle a 4-min period at 95.5”C to allow the double-stranded tem-

plate to denature,

and the last cycle included

an b-min period

A ~-PI portion of the mixture was analyzed by agarose-gel sis, and the DNA was purified with GeneClean (BiolOl.

at 77’C.

electrophoreLa Jolla, CA,

USA). Site-directed mutagenesis was performed (Kunkel et al.. 1987) using the protocol provided in the MutaGen kit (Bio-Rad) on ssDNA obtained from appropriate clones in pBluescript SK(+) (Stratagene). Mutagenic

primers

were phosphorylated

The RAM1

pMPM16-2. The ORF for RAMI in pT7-DPRI was reconstructed by PCR primer mutagenesis to introduce an NdeI site in the start codon, an EcoRI site that overlapped the NdeI sequence by I bp, and an XhoI

pMPM9-1.2

CA, USA) and used in site-directed

kinase.

cloned

RAM2 RAM1

and MVI 190 (ung+, dut+)

with T4-polynucleotide

0RF3

ORF2

RAM1

pMPM7- 1 pMPM16-2

or enzymatically

ORF in pMPMS-1 was cloned into pARC306N as a 0.77-kb NdelXhoI fragment to give pMPM9-1. The RAM2 ORF in pMPM5-2 was

of Ram1 and Ram2 protein

chemically

during

solid-phase

the RAM2IRAMl and RAMIIRAM2 junctions coupled by overlapping stop and start codons, as described for pMPMll-l/2 and pMPM13-2/l. Cell-free homogenates from pARC306N (control), pMPM7-1, and pMPM16-2 were inactive. No significant activity was seen when samples from the pMPM7-1 and pMPM 16-2 strains were mixed. SDS-PAGE analysis of the clarified supernatant and pellet fractions from JMlOl[pMPM7-1) and JMlOl[pMPM16-21 indicated that most of the Ram1 (43 kDa) and Ram2 (34 kDa) protein had precipitated in inclusion bodies (data not shown). In contrast, PFTase activity was found in all of the transformants with synthetic operons containing RAM1 and RAM2. The results are summarized in Table III. Cellfree homogenates from JMlOl[pMPM9-1,2], where the ORFs were separated by a 20-nt spacer containing an AGGAG RBS 5 bp upstream from the start codon in RAM2, had low levels of activity. The PFTase activity decreased twofold when the spacer was removed and the stop codon for Ram1 was fused to the start codon for Ram2 in pMPM 11 -l/2. However, switching the positions of RAMI and RAM2 in pMPM13-2/l gave a tenfold increase in PFTase activity. Although initial assays

as a 2.09-kb

&I-Hind111

from the TAA stop codon.

The RAMZ

ORF was sequenced,

fragment

to give

The gel-purified

and comparisons

frag-

with the

sequence (Goodman et al.. 1988) indicated a dT-+dA point that resulted in a V135D mutation in the Ram1 protein. The

PCR experiment was repeated with the same result. Examination of the original sequencing gels (Goodman et al., 1988) revealed a typographical error in the published sequence. The wt Ram1 has an AsP’~~. Similar PCR experiments

were used to introduce

NdeI and PstI restric-

tion sites at the ATG start codon and just downstream from the TAA stop codon. respectively, in RAM2 in pBH57 (He et al., 1991). The 0.96kb PCR fragment

was cloned

between

the PstI and Hind111 sites of

pBluescript-SK(+) to give pMPMS-2m. The RAM? insert contained a silent dT+dC mutation in the Thrs5 codon and a dA+dG change that produced

an E260G

mutation

in the Ram2

protein.

The mutations

found in pMPMS-2m were also present in the sequence of RAM2 in pBH57 synthesized from genomic DNA by PCR. In this case, a review of the original sequencing data indicated that the published sequence for RAM2 (He et al., 1991) was correct. Site-directed mutagenesis was used to correct the mutation at codon 260 and restore the Glu residue found in the wt protein.

In addition,

a silent TCA to TCT mutation

in

the Se? codon removed a NdeI restriction site, and a silent ATA+ATT mutation in the Ile6’ codon removed a second natural NdeI site. Finally, a CCC to CCG point mutation switched the Prozh3 to a more common E. co/i usage (Sharp et al.. 1988). The modified ORFs in plasmids pMPMS-1 expression systems.

and

pMPM5-2

were

used

to construct

the

indicated that the addition of a second copy of RAM2 behind RAMI in pMPM14-2/l/2 produced even higher levels of PFTase activity than the other constructs, a more careful examination showed that the activity in JMlOl[pMPM14-2/l/2] was slightly lower than in JMlOl[pMPM13-2/l]. SDS-PAGE was performed on cell-free homogenates from each of the transformants. Ram1 was the more abundant protein in pMPM9-1,2 and pMPMll-l/2. There was no clearly discernable band of 34 kDa for Ram2. Gels from pMPM 13-2/ 1 and pMPM 14-2/ l/2 indicated that the relative amounts of both proteins were more closely balanced. Several attempts were made to purify wt yeast PFTase to homogeneity directly from S. cereuisiae and from E. coli pMPM13-2/l and pMPM14-2/l/2 transformants by affinity chromatography using a variety of peptide ligands based on CAAX sequences in farnesylated yeast proteins or in sequences recognized by mammalian PFTase. These included ACVIA, CCIIS, KTSCVIM, KTSCVIA, and full-length unmodified a-mating factor peptide (Clarke, 1992). Although it was possible to bind the enzyme to KTSCVIM, KTSCVIA, and unmodified

L

AAC

N

AAC

CGA R

CGA R

CGA R

CGA R

GAG E

Q

CAG

CAG Q

Q

CAG

Q

CAG

GAG E

AGA R

AFA R

R

AGA

ACA R

TAC Y

CTCGACCTGCAGGAGTACAT

A-XC M

GAG E

GAG E

TAC Y

P CCA

P CCA Q CAG

Q GAG

f)RF2/0RF3b

s

E GAG

E GAG

TCT

P

E GAG

E GAG

CCA

s

F TTT

F ‘ITT

AGT

TAATG M

dog

stop TAATG M stop TAATG M

GAG E

GAG E

GAG E

GAG E

GAG E

GAG E

TAC Y

TAC Y

TAC Y

“Plasmid pMPM9-1,2 was constructed by cloning the 1.04kb PstI-PvuII fragment from pMPM&2, which contained the AGGAG BBS, RAM.& and stop codon, between the PstI and EcoRV sites of pBlaeseript SK-(i) to give pMPM7-2, Then, the 1.61-kb Psrf-@XII fragment from pMPM6-1 was ligated with the 3.55kb PstI-ABIXXfragment from pMPM%2 to give pMPMPf,Z The two QREs were coupled by deletion mutagenesis (Kuakel et al., 1987) with the antisense strand primer (3’-GTG TTT TAT TTA TTA GAC TTG ACTAC GCT GTC TCT CAT CCT TCC AGG-5’) which overlapped the TGA translation termination codon of RAM1 (complementary triplet is underlined) with the ATG start codon of RAM2 (complementary is in bold), to generate pMI?Mll-l/2. The RAM1 and RAM2 structural genes were transposed as follows. First, pMPMS-1 was mutated using the antisense strand primer (3’~CC CGA CGT CCT TAG CTA TAC GCT GCT TCT-5’) to introduce a CM site {bald) before the start eodon in the RAMI ORF. The t.31-kb CUKpnf fragment from the Chf mutant wiui then cloned between the ClaI and Kpnl sites of pMPMS-2, f&owed by deletion mutagenesis with the antisen8e strand primer (3’~GG TTA AAT ACA AGA ECxT TCA ATTAC CTC CTC ATG CTA ATA AGT CTG-S’), which coupled Ihe RAM2 and RAM1 ORFs to give pMPMlO2/i. Next, pMPM9-1,2 was cut with Ps~X+Nhe1, the overhanging ends were filled-in by treatment with dNTPs and Klcntrw polymerase, and the blunt ends were ligated to generate pMPM15-1. This plasmid contained RAM1 and a 0.0%kb segment of the 3’ end of RAM2 between the stop codon of RAM1 and the transcription terminator. Ligation of the 1.77&b N&I-&oE fragment from pMPMIO.2jl into the complementary site in pMPMlS-I gave pMPMI?-2/I.. Next, the 1.77-kb Nkl-Ncol fragment from pMPMKl-2J1 was cloned between the N&I and NcoE sites of pMPMl I-I/2 to generate pMPMf4-Z/f/2. Kunkel mutagenesis was used to cause an S431Q mutation and to add the tripeptide (underlined) to the Ram1 protein encoded in pMPMt4-Z/1/2 with the antisense strand primer (SAGG TTA AAT AGA AGA GGT GTG CTC CTC AAA ATTAC CTC CTC ATG CTA ATA-3’);the resulting plasmid, pGP14-2/l/2, had a strong AGGAGGAG (italicized) RBS embedded within the QEEF codons 6 nt upstream from the start codon for-& second RAM2 ORF (bold) in the ATTAC RAM.i/stop RAMZjstart sequence. The RAM&RAMI-RAM.2 cassette in pGP14-2/l/2 was cIoned into pTTQlBN to place the synthetic operon under control of an IPTG-inducible tat promoter in pGPl14-2/l/Z. “RBS sqrtenees are in bold. Stop eodoxts are aligned except for pMPM9-1,2_ OR.Fs are designated in Table I.

CTG

w

pGP114-?/l/2

AAT

N

18 CTG

N

AAT

pGP14-2/l/2

A&

C!&

A&

pMPM14-2jlj2

N

AA@

L

CTG

N

AAT

TAATG M stop TGATE M stop TGATG M stop TGATG M stop TGATG M

stop

AGT

s

stop

TAA

s

AGT

CCA

P

TCT

s

P

CCA

TCT

S

QRFlJURF2b

and RAM2 ORFs

pMPMl3-211

pMPMll-l/2

pMPM9-I,2

Plasmid”

Sequences at junctions of RAM1

TABLE II

45 TABLE

III

Expression

a strong of yeast re-PFTase

RBS can also be incorporated

C-terminal

Straina

Specific activityb (pm01 mini’ mgg’)

JMlOI[pMPM7-I]

<1o-5 <10-s

JMlOl[pMPM16-21 JM101[pMPM7-1],JM101[pMPM16-2]’

JMlOl[pMPM13-2/l] JMlOl[pMPM14-2/l/2] JMlOl[pGP14-2/1,‘2] JMlOl[pGPl14-2/l/2]”

6.8 x IO_’ 3.0 x 1om2 6.8 x 1o-4

DHSa[pGPI

2.1 x 1om2

in LB containing

of the transformants

were incubated

100 pg Ap/ml. A 30-pl portion

5 bp upstream The

washed

with

IOOmM

BME. and disrupted by sonication PMSF. The cell-free homogenate

K*phosphate

at 37°C

was used to inoculate

pH

6.0/10mM

in the same buffer containing was clarified by centrifugation.

1 mM

“The cell-free homogenates and supernatants were assayed for PFTase activity (Pompliano et al., 1992) and the supernatants and pellets were analyzed by 0.1% SDS-12%PAGE and visualized with Coomassie blue R. Protein concentrations were measured by the Bradford procedure (Bradford, 1976). ‘Cells were mixed and disrupted. dCuhures were induced A mo nm - 0.6.

Cell-free homogenates

with 0.2 mM IPTG

at the C-terminal

a QEEF coupling

for immunoaffinity aa of Ram 1 with

before the stop codon

from the start codon

RAMZ-RAMl-RAM2

The

overnight

buffer

epitope

sequence

under control of an IPTG-inducible pGP 114-2/ l/2.

10 ml of SB (32 g of bactotryptone/20 g of yeast extract/5 g of NaCI/S ml of 1 M NaOH/lOO mg Ap. all per liter). Cells were harvested after 12 h at 37°C

a-tubulin

chromatography. A S43 1Q mutation

levels

substantially “Single colonies

an

into

translational

was con-

structed (see footnote a in Table II). The resulting plasmid, pGP14-2/l/2, had a strong RBS (AGGAGGAG)

3.8 xIO-~ 1.5 x 1o-4 1.9x10-3

14-2/l/2]”

provide

an EEF insert appended

<1o-5

JMlOl[pMPM9-1,2] JMlOl[pMPMll-l/2]

and

motif to both improve

were assayed.

(final concentration

when

a-mating factor peptides at low salt concentrations, interactions between the enzyme and the affinity ligand were not sufficiently specific to allow purification of PFTase from other proteins bound to the column. Gomez et al. (1993) reported similar problems in their attempts to purify yeast PFTase by affinity chromatography. The lack of success for yeast PFTase versus protein prenyltransferases from mammalian sources (Reiss et al., 1990; Moores et al., 1991; Yokoyama and Gelb, 1993) can be attributed to a weaker binding of protein substrates, as reflected in substantially higher Michaelis constants for the yeast enzyme (see section c). The modest levels of expression and lack of success in finding a suitable affinity column for the purification of yeast PFTase based on substrate binding prompted us to consider another approach. The recent purification of proteins containing an EEF C-terminal a-tubulin epitope by immunoaffinity chromatography (Stammers et al., 199 1) using immobilized, commercially available monoclonal YL1/2 antibodies presented an interesting solution. The trpE protein in E. coli has a C-terminal QETF tetrapeptide sequence, and the nt sequence at the translationally coupled trpE/trpD junction in the trp operon 5’-CAG GAG ACT TTC TGATG contains an RBS (bold) within the codons for Gln and Glu (Yanofsky and Crawford, 1987). With the proper choice of codons,

translationally

of

prenyltransferase

in E. coli transformants coupled

RAMl/RAMZ

of the RAM2 was tat

also

unit. placed

promoter

activity

increased

containing motif

in

the

patterned

after the E. coli trpE/trpD junction. As shown in Table III, the specific activity of re-PFTase::QEEF in JMlOl[pGP14-2/l/2] was 3 x10-’ umol min-’ mgg’. The enzyme activity was 40-fold lower in the IPTGinduced JMlOl[pGPl14-2/l/2]. However, E. coli DHSa[pGPl14-2/l/2] produced the same levels of yeast re-PFTase::QEEF as JMlOl[pGP14-2/l/2]. Our E. coli systems gave approx. 120-fold higher levels of activity than recently reported by Gomez et al. ( 1993) for wt yeast PFTase in a co-transformed yeast strain. (c) Purification of yeast re-PFTase::QEEF The re-PFTase::QEEF was purified from induced cells of E. coli DHSa[pGPl14-2/l/2]. Upon chromatography on DE52 with a step gradient, re-PFTase::QEEF eluted just behind the major protein peak in the 200 mM NaCl step as shown in Fig. 1A (see legend for details). This material was loaded directly onto an immunoaffinity column, prepared by crosslinking monoclonal YL1/2 atubulin antibodies to Protein G-Sepharose, and rePFTase::QEEF eluted at 4°C as a single sharp peak with 5 mM Asp-Phe (Fig. 1B). The purified enzyme was obtained in an overall yield of 25% with a 25-fold purification. A gel from SDS-PAGE of samples at each stage of the purification is shown in Fig. 1C. From the intensity of the bands, we estimate that re-PFTase::QEEF was greater than 90% pure. Purified re-PFTase::QEEF was stored in elution buffer containing 40% glycerol at - 70°C until needed. Under these conditions the enzyme lost < 5% of its activity during two freeze-thaw cycles. Michaelis constants for re-PFTase::QEEF were estimated from curve fitting to hyperbolic plots (Enzfitter; Sigma, St. Louis, MO, USA) of initial velocity versus substrate concentration in the presence of a fixed concentration of the second substrate. The values we obtained for V,,, = 0.53 umol/min per mg (k,,, =0.7/s) and KEp = 5.5 uM are similar to those reported by Gomez et al. (1993) for enzyme from their overproducing yeast strain.

46

C

B -

--c

Absorbance Activity

kDa

Absorbance Activity

200 97 -

-

66

43 -

Ram1

29

Ram2

-

0.1 0.0 100

0

200

300

400

0

10

20

16.

30

Volume (mL)

14.

Fig. 1. Purification of yeast re-PFTase::QEEF. A 1.5-g portion of wet cells was disrupted BME buffer containing 1 mM PMSF. The clarified cell-free extract was dialyzed against

by sonication two changes

in 40 ml of 50 mM TrisHCl of 2 liters each of disruption

pH 7.0/5 mM buffer at 4°C

67 mg of protein, specific activity =0.021 umol min- ’ mg- I. (A) Chromatography on DE52 at 4°C. The dialysate was loaded onto a 3 x 12 cm column of DE52, previously equilibrated with 50 mM TrisHCl pH 7.0/50 uM ZnCl,/S mM MgCI,/lO mM BME. Protein was eluted with a stepwise gradient of 0 mM, 100 mM, 200 mM, and 1 M NaCl in the same buffer, and fractions were assayed for PFTase activity. PFTase eluted at 200 mM NaCl (12 mg of protein, Chromatography IgG2a containing

specific activity =0.068 pm01 min- ’ mgg’). Elution: Step gradient with A (100 mM NaCl), B (200 mM NaCl), C (I M NaCl). (B) on an a-tubulin immunoaffinity column at 4°C. An anti-a-tubulin affinity matrix was prepared as follows from rat ascites fluid a-tubulin monoclonal antibodies (Serotec, Oxford, UK). GammaBind G (Pharmacia, Alameda, CA, USA; 2 ml of settled resin) was

washed with 10 ml of 10 mM Naphosphate pH 7.0/10 mM EDTAjl50 mM NaCl (binding buffer) incubated with end-over-end rotation at 4°C with 1.4 ml of ascites fluid in 8 ml of binding buffer for 1 h and washed twice with 0.2 M Na,B,O, pH 8.9. The resin was suspended in 10 ml of borate buffer, and 70 mg of dimethyl pimelimidate (final concentration of 25 mM) was added to crosslink the antigen-antibody complex. After rotational mixing for 45 min at 22°C the solution was decanted, and the resin was incubated with 0.2 M ethanolamine pH 9.0 for 1 h at 22°C. The supernatant was decanted,

and the resin was washed

with binding

buffer and then stored

at 4°C in binding

buffer containing

0.05%

thimerosal.

A 5 x 40 mm

glass column was gravity packed with the anti-m-tubulin support and equilibrated with binding buffer containing 10 mM BME at a flow rate of 2-4 ml/h. DE52-purified PFTase (9-10 ml) was loaded onto the column at 2 ml/h. The resin was washed with 4-6 ml of binding buffer/BME, and re-PFTase::QEEF activity was eluted with binding buffer/BME containing 5 mM Asp-Phe, 0.85 mg of protein, specific activity=0.53 umol min-’ mg -I. A=loading of DE52-purified material; B=wash with 10 mM Naphosphate pH 7.0/10 mM EDTA/150 mM NaCI/lO mM BME; C= elute with 10 mM Na.phosphate pH 7.0/10 mM EDTA/150 mM NaCl/S mM Asp-Phe/lO mM BME. (C) Analysis of samples from the purification of yeast re-PFTase::QEEF by 0.1% SDS-12%PAGE. Lanes: 1, size markers (in kDa): 200, myosin H-chain; 97, phosphorylase b, 68, bovine serum albumin; 43, ovalbumin; 29, carbonic anhydrase; 18, S-lactoglobin; 14, lysozyme; 2, cell-free homogenate; 3, supernatant after a low-speed spin; 4, after DE52 chromatography; 5, after immunoaffinity chromatography. The gel was stained with Coomassie blue R.

Although there is no published KM for H,,,-PACVIA, our value of K~s-PACV’A= 15 uM is similar to that reported by Gomez et al. (1993) for farnesylation of GST-CTTS, a glutathione-S-transferase fusion protein with a yeast Rasf C-terminal tail (Clarke, 1992). (d) Conclusions

Farnesylation of proteins in S. cereuisiae is mediated by PFTase. The enzyme is a heterodimer consisting of a 43-kDa subunit encoded by RAM1 and a 34-kDa subunit encoded by RAMZ. Of the plasmids evaluated for their ability to synthesize yeast PFTase directly in E. coli, maximal levels of activity were found for JMlOl[pGP142/ l/2] and DHSa[ pGPl14-2/l/2]. These plasmids contained synthetic operons with a RAMZIRAMlIRAMZ motif and translationally coupled RAMIIRAM2 unit under control of either a rec7 or a tat promoter. The translation termination codon of RAM1 was fused to the translation initiation codon of RAMZ, and the 3’ end of the RAM1 ORF was mutated to imbed an RBS sequence

(AGGAGGAG) 5 bp upstream from the Met start codon of RAM2. The re-Ram1 protein had a C-terminal QEEF sequence that facilitated purification of the re-enzyme by immunoaffinity chromatography on an anti-a-tubulin column. Heterodimeric yeast re-PFTase::QEEF constituted approx. 4% of total cellular protein in early stationary phase cultures of JMlOl[pGP14-2/l/2] or IPTGinduced cultures of DHSa[pGP114-2/l/2]. The reenzyme was purified 25-fold to >90% homogeneity by ion-exchange and immunoaffinity chromatography, and its kinetic constants were similar to those previously reported for the wt heterodimer. The QEEF motif in combination with fused stop and start codons appears to be a useful construct for coupling translation of ORFs in a synthetic operon. In addition, the presence of the a-tubulin epitope in one of the gene products provides a convenient handle for the rapid purification of the re-protein by immunoaffinity chromatography. The QEEF sequence should be useful for the rapid purification of proteins containing site-directed mut-

47 ations for kinetic studies and should be generally applicable in a wide variety of situations.

He, B., Chen, P., Chen, S.-Y., Vancura, S.: RAM2,

an essential

polypeptide

components

K.L., Michaelis,

gene of yeast, and RAM1 of the farnesyltransferase

We wish to thank J. Rine for samples of pT7-DPRl and pT7-DPRl/H,,, and S. Powers for pBH57. We also thank R. Khosravi-Far and C. Der for helpful advice on the purification of H,,, protein. Oligodeoxyribonucleotides and peptides were synthesized and purified by Dr. R. Schackmann, Utah Regional Cancer Center Protein/DNA Core Facility. M.P.M. was a Deutsche Forschungsgemeinschaft Fellow. G.D.P. was a NIH Senior Fellow of the National Heart, Lung, and Blood Institute, HL-08585. J.M.D. was a NIH Postdoctoral Fellow, National Institute of General Medical Sciences, GM-15286. This work was supported by NTH grant GM-21328.

specific mutagenesis without Enzymol. 154 (1987) 367-382.

phenotypic

Moores,

Bradford, M.M.: A rapid and sensitive method micrograms of protein utilizing the principle

for the quantitation of of protein-dye binding.

72 (1976) 248-254.

Clarke, S.: Protein isoprenylation and methylation at C-terminal ine residues. Annu. Rev. Biochem. 61 (1992) 355-386. Der, C.J., Krontiris, T.G. and Cooper, human bladder and lung carcinoma

cyste-

G.M.: Transforming genes of cell lines are homologous to

and Kirstin sarcoma viruses. Proc. Natl. 3637-3640. Tripathy, S.K., O’Rourke, E., Manne, V. yeast protein farnesyltransferase is strucsimilar to its mammalian counterpart.

Biochem. J. 289 (1993) 25-31. Goodman, L.E., Perou, C.M., Fujiyama, A. and Tamanoi, F.: Structure and expression of yeast DPRZ, a gene essential for the processing and intracellular localization of ras proteins. Yeast 4 (1988) 271-281.

Methods

S.L., Schaber,

M.D., Mosser,

SD.,

Rands,

E., O’Hara,

M.B..

Garsky, V.M., Marshall, M.S., Pomphano, D.L. and Gibbs, J.B.: Sequence dependence of protein isoprenylation. J. Biol. Chem. 266 (1991) 14603-14610. Olins, P.O., Devine, C.S., Rangwala, S.H., and Kavka, K.S.: The T7 phage gene 10 leader RNA, a ribosome-binding site that dramatically enhances the expression of foreign genes in Escherichin coli. Gene 73 (1988) 227-235. Pompliano, D.L., Rands, E., Schaber, M.D.. Mosser, S.D., Anthony, N.J. and Gibbs, J.B.: Steady-state mechanism of ras farnesyhprotein transferase. Biochemistry 3 1 (1992) 3800-3807. Reiss, Y., Seabra,

Sanger,

REFERENCES

selection.

Manne, V., Bekesi, E. and Kung, H.-F.: Ha-r-as proteins exhibit GTPase activity: point mutations that activate Ha-ras gene products result in decreased GTPase activity. Proc. Natl. Acad. Sci. USA 82 (1985) 376-380.

M.C., Goldstein,

J.L. and Brown,

of ras farnesyl: protein transferase. Methods Enzymol. 1 (1990) 241-245.

the ras genes of Harvey Acad. Sci. USA 79 (1982) Gomez, R., Goodman, L.E., and Tomanoi, F.: Purified turally and functionally

the two

that prenylates

a-factor and Ras protein. Proc. Natl. Acad. Sci. USA 88 (1991) 11373-I 1377. Kunkel, T.A., Roberts, J.D. and Zakour, R.A.: Rapid and efficient site-

ACKNOWLEDGEMENTS

Anal. Biochem.

S. and Powers, encode

F., Nicklen,

terminating 5463-5467. Sharp,

S. and Coulson,

inhibitors.

Proc.

P.M., Cowe, E., Higgins,

Methods:

M.S.: Purification Companion

A.R.: DNA sequencing Natl.

Acad.

Sci. USA

to

with chain74 (1977)

D.G., Shields, D.C., Wolfe, K.H. and

Wright, F.: Codon usage patterns in Escherichia cd, Bacillus subtilis, Saccharomyces cerevisiae, Schizosaccharomyces pombe. Drosophila melanogaster, and Homo sapiens: a review of considerable within-species diversity. Nucleic Acids Res. 16 (1988) 8207-8211. Stammers, D.K., Tisdale, M., Court, S., Parmar, V., Bradley, Ross, C.K.: Rapid purification and characterization of HIV-l

C. and reverse

transcriptase and RNaseH engineered to incorproate a C-terminal tripeptide a-tubulin epitope. FEBS Lett. 283 (1991) 298-302. Yanofsky. C. and Crawford, I.P.: The tryptophan operon. In: Neidhardt, F.C., Ingraham, J.L., Low, K.B., Magasanik, B., Schaechter, M. and Umbarger, H.E. (Eds.), Escherichia co/i and Salmonella typhimurium. Cellular and Molecular Biology, Vol. II. American Society for Microbiology, Washington, DC, 1987, pp. 1453-1472. Yokoyama, K. and Gelb, geranylgeranyltransferase.

M.H.: Purification of a mammalian protein J. Biol. Chem. 268 (1993) 4055-4060.