FEMS Microbiology Letters 222 (2003) 237^242

www.fems-microbiology.org

Expression and biochemical characterization of the 1-HO-carotenoid

methylase CrtF from Rhodobacter capsulatus

Frank Badenhop, Sabine Steiger, Manuela Sandmann, Gerhard Sandmann
Biosynthesis Group, Botanical Institute, J.W. Goethe Universita«t, P.O. Box 111932, D-60054 Frankfurt/Main, Germany

Received 23 January 2003; received in revised form 1 April 2003; accepted 1 April 2003

First published online 24 April 2003

Abstract

In purple bacteria, acyclic 1-methoxy carotenoids like spheroidene or spirilloxanthin are essential components of the photosynthetic
apparatus. One of the last steps of their biosynthesis involves O-methylation of the 1-hydroxy group. The 1-HO-carotenoid methylase
CrtF from Rhodobacter capsulatus catalyzing this type of reaction was expressed in Escherichia coli in an active form. It was then purified
by affinity chromatography and biochemically characterized. The enzymatic reaction depends on S-adenosylmethionine as the only
cofactor. By complementation in E. coli, the substrate specificity of the enzyme was determined. It could be shown that the enzyme
converts not only all possible 1-hydroxy carotenoids in the spheroidene/1P-HO-spheroidene biosynthetic pathway of R. capsulatus but also
carotenoid intermediates leading to the formation of spirilloxanthin in a pathway which is absent in R. capsulatus but present in related
species.
1 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Keywords : Acyclic 1-HO-carotenoid methylase; CrtF; Rhodobacter capsulatus; Spheroidene biosynthesis ; Spirilloxanthin biosynthesis

1. Introduction agent of the 1-hydroxy group of spheroidene [6,7]. How-

ever, detailed functionality studies on this enzyme includ-
In photosynthetic bacteria, carotenoids are essential ing in vitro reactions are lacking to date.
components of the photosynthetic intercytoplasmic mem- Since the gene crtF encoding the 1-HO-carotenoid meth-
brane. The dominating carotenoids in anaerobic cultures ylase from R. capsulatus has been cloned and functionally
of purple bacteria are those with acyclic end groups carry- identi¢ed [6,8], it is available for heterologous protein
ing a 1-methoxy substituent [1,2]. In the biosynthetic path- production. In this investigation, the 1-HO-carotenoid
way, these carotenoids are derived either from neurospor- O-methylase was expressed, a new in vitro assay system
ene or from lycopene. The subsequent reactions involved developed and the catalytic properties of the enzyme de-
are 3,4-desaturation and 1,2-hydration [1,3]. Methylation termined.
of the 1-hydroxy group is one of the last steps in the
formation of acyclic carotenoids like spheroidene and spi-
rilloxanthin. From the enzymes catalyzing the formation 2. Materials and methods
of both carotenoids, only the 3,4-desaturases from Rhodo-
bacter sphaeroides and Rubrivivax gelatinosus have been 2.1. Plasmids, bacterial strains and growth conditions
puri¢ed and biochemically characterized [4,5]. Little is
known about the enzymatic properties of the O-methylase. Construction of plasmid pUC18crtF was previously de-
Labelling studies of Rhodobacter capsulatus cells with me- scribed [4]. From this plasmid, pRKcrtF was derived by
thionine and R. sphaeroides with S-adenosylmethionine partial digestion with PvuII and ligation of the 1.5-kb
indicated that the latter compound is the methylating fragment into the HindIII-digested, Klenow-treated and
dephosphorylated vector pRK404 [9]. The pPQEcrtF-
overexpressing plasmid used for protein puri¢cation was
* Corresponding author. Tel. : +49 (69) 79824730;
obtained by KpnI/PstI digestion of pUCcrtF and ligation
Fax : +49 (69) 79824822. of crtF into the PstI/KpnI site of pPQE-31 [10] which is a
E-mail address : [email protected] (G. Sandmann). modi¢cation of the pQE-31 vector (Qiagen, Hilden, Ger-
0378-1097 / 03 / $22.00 1 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
doi:10.1016/S0378-1097(03)00302-1

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238 F. Badenhop et al. / FEMS Microbiology Letters 222 (2003) 237^242

many). Plasmid pUC19K8 with an expressing metK gene were harvested by centrifugation at 6000Ug for 15 min
from Escherichia coli was constructed by restriction diges- at 4‡C, resuspended 1:10 in 0.2 M potassium phosphate
tion of pK8 [11] using PstI/EcoRI and ligation into the bu¡er pH 8.0 containing 15% glycerol and 0.2 M NaCl
same sites of pUC19 [12]. and broken in a French pressure cell at 95 MPa. After
The E. coli strain M15 harboring plasmid pREP4 (Qia- incubation of 5 ml of the supernatant with 750 Wl of the
gen) was the host for pPQEcrtF with the 1-HO-carotenoid Talon resin (BD Clontech, Heidelberg, Germany) for
O-methylase gene crtF from R. capsulatus [8] under the 1^1.5 h on ice, the mixture was packed in a column and
control of the lacZ promoter and a N-terminal six-histi- non-bound proteins were eluted by washing four times
dine sequence used for a⁄nity puri¢cation. For protein with 1.5 ml of the above bu¡er containing 10 mM imida-
overexpression, the culture was cultivated at 37‡C in Lu- zole. For elution of proteins (in 1.5-ml fractions) increas-
ria^Bertani broth in the presence of ampicillin (100 Wg ing imidazole concentrations were used. Fractions used for
ml31 ) and kanamycin (25 Wg ml31 ) until it reached an in vitro assays were desalted on Sephadex G25 (PD 10
optical density at 600 nm of 0.1. Then, 0.5 mM of isopro- column, Amersham, Freiburg, Germany). For the docu-
pyl L-D-thiogalactopyranoside was added and the culture mentation of the di¡erent puri¢cation steps 270 Wl of each
was grown overnight at 26‡C. fraction was precipitated after addition of 6% trichloro-
acetic acid and applied for separation on a 12.5% sodium
2.2. Complementation experiments and substrate production dodecyl sulfate^polyacrylamide gel (SDS^PAG) [16]. The
relative amounts of the expressed proteins were estimated
For complementation experiments, E. coli JM101 was after separation on the SDS^PAG by staining with Coo-
used as a host and di¡erent carotenoid backgrounds were massie brilliant blue and scanning using a densitometric
established by transformation with various carotenogenic software. Protein concentrations were determined with the
plasmids. Details of their structure and function are given Bio-Rad protein assay.
in [13]. For analysis of substrate speci¢city of CrtF, these
transformants were co-transformed with either pPQEcrtF 2.4. In vitro enzyme assay and analysis of the reaction
or pRKcrtF. Growth was for 2 days at 28‡C in the pres- products
ence of appropriate antibiotics (i.e. ampicillin 100 Wg ml31 ,
chloramphenicol 35 Wg ml31 and tetracycline 25 Wg ml31 ) The assays contained 250 Wl of the di¡erent puri¢cation
according to the plasmids used. Analysis of the carote- fractions of the O-methylase (equivalent to 2^5 Wg pro-
noids formed by CrtF was performed by high performance tein), 50 Wl hydroxydemethylspheroidene, 150 Wl homoge-
liquid chromatography (HPLC) on a Nucleosil C18 3-Wm nate of broken JM101/pUC19K8 cells with 50 Wl of cofac-
column eluted isocratically with acetonitrile/methanol/ tor mix containing methionine 0.1 M, ATP 0.13 M and
2-propanol (50:40:10, v/v) at a £ow rate of 1 ml min31 . MgCl2 0.26 M [17] or alternatively 50 Wl of S-adenosylme-
The separated carotenoids were detected with a Kontron thionine 0.1 M in a ¢nal volume of 1 ml 0.2 M potassium
440 diode array detector and spectra were directly re- phosphate bu¡er pH 8.0 containing 15% glycerol and
corded on-line. Identi¢cation of carotenoids formed by 0.2 M NaCl. After incubation overnight at 28‡C, the re-
function of CrtF was by co-chromatography with authen- action was terminated by addition of 2.5 ml methanol.
tic standards and/or spectral characteristics as well as The remaining substrate and the products formed were
chromatographic behavior. Spheroidene and 1P-HO-spher- extracted from the incubation mixture with diethyl ether/
oidene were isolated from R. gelatinosus wild-type and petroleum ether (b.p. 35^60‡C) (1:9, v/v) and analysed by
CH3 O-neurosporene and CH3 O-lycopene from the SID HPLC as described above.
mutant [5]. For determination of the Km value for hydroxydeme-
Hydroxydemethylspheroidene applied as substrate in thylspheroidene with the 1-HO-carotenoid O-methylase,
the in vitro reaction was extracted from freeze-dried cells substrate concentrations in the range from 2 to 40 WM
of the E. coli transformants carrying plasmids pACCRT- were used. Km and Vmax values were obtained from double
EBIR:c: +pSO50 [13] with methanol containing 6% KOH, reciprocal Lineweaver^Burk plots of the reaction rates ver-
by heating for 20 min at 60‡C and partitioned in 10% sus the substrate concentration with ¢ve data points and a
diethyl ether in petrol. For further puri¢cation, hydroxy- correlation coe⁄cient for linearity of s 0.97.
demethylspheroidene was separated by chromatography
on alumina columns [14]. Quantitation of hydroxydeme-
thylspheroidene and hydroxyspheroidene was carried out 3. Results and discussion
using the extinction coe⁄cient for spheroidene [15] which
contains an identical chromophore. 3.1. Substrate speci¢city of CrtF determined by genetic
complementation in E. coli
2.3. Enzyme puri¢cation
Many carotenogenic enzymes possess a broad substrate
The overnight induced cells (M15/pREP4+pPQEcrtF) speci¢city. This feature allows alternative pathways to op-

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 F. Badenhop et al. / FEMS Microbiology Letters 222 (2003) 237^242 239

erate when other enzymes of the pathway are inactivated dition to 1-HO-neurosporene (peak 2) and neurosporene
[18]. For the O-methylase substrate speci¢city was tested (peak 4). 1P-HO-demethylspheroidene (trace B, peak 5)
by genetic complementation in E. coli. For this purpose, and demethylspheroidene (peak 7) are methylated to
E. coli was co-transformed with a plasmid mediating 1P-HO-spheroidene (peak 6) and spheroidene (peak 8), re-
the formation of the desired substrates together with spectively. In trace C, 1-HO-lycopene (peak 11) and
pPQEcrtF expressing the O-methylase (Fig. 1). Under con- 1,1P-(HO)2 -lycopene (peak 9) are produced as substrates.
ditions when hydroxyneurosporene derivatives are formed, The methoxy derivatives formed from them are 1-CH3 O-
1-CH3 O-neurosporene (trace A, peak 3) and 1-CH3 O-1P- lycopene (peak 12) and 1-CH3 O-1P-HO-lycopene (peak
HO-neurosporene (peak 1) were detected by HPLC in ad- 10). Complementation with plasmids pACCRT-EBIE:u: +
pSO50 [5] resulted in the formation of 1,1P-(HO)2 -3,4,
3P,4P-tetradehydrolycopene (peak 14) and 1-HO-3,4,-dide-
hydrolycopene (peak 17). In the presence of pPQEcrtF,
the following methoxy carotenoids could be identi¢ed by
co-chromatography with the corresponding carotenoids
isolated from R. gelatinosus wild-type and SID2 mutant
[5]: 1-CH3 O-1P-HO-3,4,3P,4P-tetradehydrolycopene (peak
15), 1P-HO-spheroidene (peak16), spirilloxanthin (peak
18) and 1-CH3 O-3,4-didehydrolycopene (peak 19). All
products of the 1-HO-carotenoid methylase which are ab-
sent in controls lacking pPQEcrtF are marked in the
HPLC diagrams of Fig. 1 with underlined numbers.
In Fig. 2A all possible methylation reactions in the bio-
synthetic pathway to the carotenoid end products 1P-HO-
spheroidene and/or spheroidene depending on individual
strains of R. capsulatus [1] are outlined. Although CrtF
catalyzes all these O-methylations, only two of the reac-
tions using demethylspheroidene and 1P-HO-demethyl-
spheroidene as substrates are involved in the pathway to
the end product 1P-HO-spheroidene. The methylation
product 1-CH3 O-neurosporene (and in analogy 1-CH3 O-
1P-HO-neurosporene) represents a dead end in the path-
way since it is not further desaturated at position 3,4 [4].
All methylation reactions of Fig. 2B with acyclic hy-
droxycarotenoids as substrates unrelated to the caroteno-
genic pathway in R. capsulatus can be catalyzed by CrtF.
Among them are mono- and dihydroxycarotenoids with

6
Fig. 1. Functionality of the 1-HO-carotenoid O-methylase CrtF. HPLC
diagram of carotenoids formed by in vivo expression of pPQEcrtF in
Escherichia coli with a 1-HO-neurosporene and 1,1P-(HO)2 -neurosporene
background by additional plasmids pACCRT-EBIR:c: +pRKcrtC [4] (A),
a demethylspheroidene and 1P-HO-demethylspheroidene background by
additional plasmids pACCRT-EBIR:c: +pSO50 [5] (B), a 1-HO-lycopene
and 1,1P-(HO)2 -lycopene and background by additional plasmids
pACCRT-EBIE:u: +pRKcrtC [4] (C), or a 1,1P-(HO)2 -3,4,3P,4P-tetradehy-
drolycopene and 1-HO-3,4,-didehydrolycopene background by pACCRT-
EBIE:u: +pSO50 [5] (D), all co-transformed with pPQEcrtF. Trace E
shows an HPLC separation of an in vitro reaction of puri¢ed CrtF with
1P-HO-demethylspheroidene as substrate. The individual peaks were
identi¢ed as: 1, 1-CH3 O-1P-HO-neurosporene; 2, 1-HO-neurosporene ;
3, 1-CH3 O-neurosporene; 4, neurosporene; 5, 1-HO-demethylspheroi-
dene ; 6, 1P-HO-spheroidene; 7, demethylspheroidene; 8, spheroidene ;
9, 1,1P-(HO)2 -lycopene ; 10, 1-CH3 O-1P-HO-lycopene ; 11, 1-HO-lyco-
pene ; 12, 1-CH3 O-lycopene; 13, lycopene ; 14, 1,1P-(HO)2 -3,4,3P,4P-tetra-
dehydrolycopene; 15, 1-CH3 O-1P-HO-3,4,3P,4P-tetradehydrolycopene; 16,
1P-HO-spheroidene; 17, 1-HO-3,4,-didehydrolycopene ; 18, spirilloxan-
thin; and 19, 1-CH3 O-3,4-didehydrolycopene.

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Fig. 2. Substrate conversion by the 1-HO-carotenoid O-methylase CrtF. A: Reactions catalyzed in the biosynthetic pathway leading to the formation of
spheroidene and hydroxyspheroidene. B: Conversion reactions of hydroxycarotenoids not involved in the carotenogenic pathway of R. capsulatus. Caro-
tenoids not O-methylated by CrtF were 1P-HO-3,4-didehydrolycopene, 1-HO-j-carotene, 1,1P-(HO)2 -j-carotene and zeaxanthin.

or without a 3,4-double bond. Hydroxycarotenoids not tus, R. rubrum possesses an analogous pathway via lyco-
accepted by CrtF were 1P-HO-3,4-didehydrolycopene, pene to spirilloxanthin and in R. gelatinosus both
1-HO-j-carotene, 1,1P-(HO)2 -j-carotene both without the biosynthetic branches are operative. In all three species
7,8- and 7P,8P-double bond and cyclic zeaxanthin. From all similar enzymes are involved [2]. But comparing the en-
these complementation results the structural requirements zymes of the Rhodobacter species with those of R. gelati-
of a substrate for CrtF can be established. The 1-HO-ca- nosus with respect to their functionality, the carotene
rotenoid should be acyclic with a 5,6-double bond in con- 3,4-desaturases CrtD and the carotene hydratases CrtC
jugation with the central polyene chain. A 3,4-double di¡er in their substrate and product spectra being adapted
bond is optional. Also the other end group can be quite to an exclusive spheroidene pathway or to an additional
variable without a negative impact on methylation, pro- spirilloxanthin pathway [19]. However, the broad substrate
vided that it is not a fully desaturated i-end group. speci¢city of CrtF from R. capsulatus would allow this
Compared to the carotenogenic pathway via neurospor- enzyme to catalyze all relevant methylation reactions in
ene to spheroidene and 1P-HO-spheroidene of R. capsula- both pathways.

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 F. Badenhop et al. / FEMS Microbiology Letters 222 (2003) 237^242 241

puri¢ed enzyme. Plasmid pPQEcrtF was expressed in

E. coli under conditions to minimize inclusion body for-
mation [20,21]. A prominent polypeptide with an apparent
molecular mass of 43 kDa was detected in the crtF trans-
formant (Fig. 3, lane 3) in comparison to E. coli carrying
an empty cloning vector (lane 2). A considerable amount
of CrtF between 3 and 6% of total soluble protein was
found in the supernatant of E. coli/pPQEcrtF homogenate
after centrifugation (Table 1, Fig. 3, lane 4). In a one-step
puri¢cation by cobalt chelate a⁄nity chromatography us-
ing Talon resin, CrtF was enriched to 94% purity (Table 1)
in the 200 mM imidazole elution fraction (Fig. 3, lane 6).
The speci¢c activity increased from 7 to 320 pg HO-deme-
thylspheroidene formed per mg protein and per hour. The
puri¢ed 1-HO-carotenoid O-methylase was enzymatically
active. In the presence of S-adenosylmethionine as cofac-
tor or when methionine and ATP together with an enzyme
supernatant from E. coli/pUC19K8 were added as a
S-adenosylmethionine-generating system, 1-HO-demethyl-
spheroidene was converted to 1-CH3 O-demethylspheroi-
dene (Fig. 1E). The optimum was found at pH 8 and a
Km value of 26.6 WM was determined for the substrate
1-HO-demethylspheroidene.
In conclusion, the 1-HO-carotenoid O-methylase was
expressed as an active enzyme, puri¢ed and enzymatically
characterized. Its broad substrate speci¢city was deter-
mined by functional complementation of the crtF gene in
E. coli. Although the enzyme originates from R. capsula-
tus, a photosynthetic purple bacterium in which only the
spheroidene branch of the carotenoid pathway is opera-
tive, this enzyme is capable of catalyzing corresponding
reactions in a spirilloxanthin pathway. This feature is dif-
Fig. 3. Polyacrylamide gel electrophoresis of the di¡erent fractions re- ferent from other carotenogenic enzymes from Rhodo-
sulting from the puri¢cation of the recombinant R. capsulatus 1-HO-ca- bacter species.
rotenoid O-methylase. Lane 1: molecular mass standard ; lane 2: E. coli
JM101/pPQE31 cells; lane 3: cell homogenate of JM101/pPQEcrtF ;
lane 4: supernatant from 3; lanes 5 and 6: elution fraction with 50 mM
(5) and 200 mM (6) imidazole. Equal amounts of fractions were applied Acknowledgements
in lanes 2^4. In lane 5 and 6 fractions were 20-fold enriched.
This work was supported by the Deutsche Forschungs-
gemeinschaft with a grant to G.S. Due thanks are ex-
3.2. Puri¢cation and biochemical properties of CrtF pressed to Dr. G.D. Markham, Fox Chase Cancer
Center, Philadelphia, PA, USA for providing plasmid
Further properties of the 1-HO-carotenoid O-methylase pK8.
were determined with the heterologously expressed and

Table 1
Puri¢cation by chelate a⁄nity chromatography and yields of 1-HO-carotenoid O-methylase
Fraction CrtF protein (mg) Purity (%) Yield (%) Speci¢c activity (pg mg31 protein h31 )
Supernatant 0.87 4.5 100 7
Non-absorbeda 0.55 5.1 63
Talon fractions
50 mM imidazole 0.11 9.2 13 15
100 mM imidazole 0.10 30.3 12 84
150 mM imidazole 0.10 77.4 11 216
200 mM imidazole 0.07 94.0 8 320
a
Protein fraction not bound by the cobalt chelate a⁄nity resin.

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References [11] Boyle, S.M., Markham, G.D., Hafner, E.W., Wright, J.M., Tabor,
H. and White Tabor, C. (1984) Expression of the cloned genes en-
[1] Schmidt, K. (1978) Biosynthesis of carotenoids. In: The Photosyn- coding the putrescine biosynthetic enzymes and methionine adenosyl-
thetic Bacteria (Clayton, R.K. and Sistrom, W.R., Eds.) pp. 729^70, transferase of Escherichia coli (speA, speB, speC and metK). Gene 30,
Plenum Press, New York. 129^136.
[2] Takaichi, S. (1999) Carotenoids and carotenogenesis in anoxygenic [12] Verdoes, J., Krubasik, P., Sandmann, G. and van Ooyen, M. (1999)
bacteria. In: The Photochemistry of Carotenoids (Frank, H.A., Isolation and functional characterisation of a novel type of caroten-
Young, A.J., and Cogdell, R.E., Eds.) pp. 39^69. Kluwer Academic oid biosynthetic gene from Xanthophyllomyces dendrorhous. Molec.
Publisher, Dordrecht. Gen. Genet. 262, 453^461.
[3] Sandmann, G. (1994) Carotenoid biosynthesis in microorganisms and [13] Sandmann, G. (2002) Combinatorial biosynthesis of carotenoids in a
plants. Eur. J. Biochem. 223, 7^24. heterologous host: A powerful approach for the biosynthesis of novel
[4] Albrecht, M., Ruther, A. and Sandmann, G. (1997) Puri¢cation and structures. ChemBioChem 3, 629^635.
biochemical characterization of a hydroxyneurosporene desaturase [14] Britton, G. (1985) General carotenoid methods. Methods Enzymol.
involved in the biosynthetic pathway of the carotenoid spheroidene 111, 113^149.
in Rhodobacter sphaeroides. J. Bacteriol. 179, 7462^7467. [15] Davis, B.H. (1976) Carotenoids. In: Biochemistry of Plant Pigments.
[5] Steiger, S., Astier, C. and Sandmann, G. (2000) Substrate speci¢city Vol. 2. (Goodwin, T.W., Ed.), pp. 38^365. Academic Press, London.
of the expressed 3,4-carotenoid desaturase from Rubrivivax gelatino- [16] Laemmli, U.K. (1979) Cleavage of structural protein during assembly
sus reveals the detailed reaction sequence to spheroidene and spiril- of the head of bacteriophage T4. Nature 227, 680^688.
loxanthin. Biochem. J. 349, 635^640. [17] Park, J., Tai, J., Roessner, C.A. and Scott, A.I. (1996) Enzymatic
[6] Scolnik, P.A., Walker, M.A. and Marrs, B.L. (1980) Biosynthesis of synthesis of S-adenosyl-L-methionine on a preparative scale. Biorg.
carotenoids derived from neurosporene in Rhodopseudomonas capsu- Med. Chem. 4, 2179^2185.
lata. J. Biol. Chem. 255, 2427^2432. [18] Komori, M., Ghosh, R., Takaichi, S., Hu, Y., Mizoguchi, T., Koya-
[7] Singh, R.K., Britton, G. and Goodwin, T.W. (1973) Carotenoid bio- ma, Y. and Kuki, M. (1998) A null lesion in the rhodopin 3,4-desa-
synthesis in Rhodopseudomonas sphaeroides. S-Adenosylmethionine as turase of Rhodospirillum rubrum unmasks a cryptic branch of the
the methylation agent in the biosynthesis of spheroidene and spher- carotenoid biosynthetic pathway. Biochemistry 37, 8987^8994.
oidenone. Biochem. J. 136, 413^419. [19] Steiger, S., Takaichi, S. and Sandmann, G. (2002) Heterologous pro-
[8] Armstrong, G.A., Alberti, M., Leach, F. and Hearst, J.E. (1989) duction of two novel acyclic carotenoids, 1,1P-dihydroxy-3,4-didehy-
Nucleotide sequence, organisation, and nature of the protein prod- drolycopene and 1-hydroxy-3,4,3P,4P- tetradehydrolycopene by com-
ucts of the carotenoid biosynthesis gene cluster of Rhodobacter cap- bination of the crtC and crtD genes from Rhodobacter and
sulatus. Mol. Gen. Genet. 216, 254^268. Rubrivivax. J. Biotechnol. 97, 51^58.
[9] Ditta, G., Schmidhauser, T., Yakobson, E., Lu, P., Liang, X.-W., [20] Blackwell, J.R. and Horgan, R. (1991) A novel strategy for produc-
Finlay, D.R., Guiney, D. and Helinski, D.R. (1985) Plasmids related tion of a highly expressed recombinant protein in an active form.
to the broad range vector, pRK290, useful for gene cloning and for FEBS Lett. 295, 10^12.
monitoring gene expression. Plasmid 13, 149^153. [21] Lucht, J.M. and Bremer, E. (1994) Adaptation of Escherichia coli to
[10] Yanisch-Perron, C., Vieira, J. and Messing, J. (1985) Improved M13 high osmolarity environments: Osmoregulation of the high-a⁄nity
phage cloning vectors and host strains : nucleotide sequences of the glycine betaine transport system ProU. FEMS Microbiol. Rev. 14,
M13mp18 and pUC19 vectors. Gene 33, 103^119. 3^20.

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