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Applied and Environmental Microbiology, February 1999, p. 787-794, Vol. 65, No. 2
Institut für Technische Biochemie,
Universität Stuttgart, Stuttgart, Germany
Received 11 September 1998/Accepted 23 November 1998
The lipase from Pseudomonas cepacia ATCC 21808 (recently reclassified as Burkholderia cepacia) is widely
used by organic chemists for enantioselective synthesis and is
manufactured from recombinant P. cepacia harboring
on a plasmid the clustered genes for lipase and its chaperone. High
levels of expression of inactive lipase (40%) in Escherichia
coli were achieved with pCYTEXP1 under the control of the
strong, temperature-inducible Lipases (triacylglycerol
acylhydrolases; EC 3.1.1.3), particularly microbial lipases, have been
widely used in the hydrolysis and transesterification of triglycerides
and in the enantioselective synthesis and hydrolysis of a variety of
esters (27, 39).
In recent years, many microbial lipase genes have been isolated,
sequenced, modified, and expressed in homologous or heterologous hosts,
such as Escherichia coli, filamentous fungi, or yeasts (3, 5, 17, 34, 37). Lipases from Pseudomonas
alcaligenes (2, 25) are extensively used as an additive
in laundry detergents, and lipases from Pseudomonas species
are widely used as catalysts in organic synthesis (12, 36).
In addition, these various Pseudomonas lipase preparations
are commercially available (e.g., Amano YS, P, and AH; Fluka SAM-II).
Among these, P. cepacia lipase preparations are the
most predominant and have often been used by organic chemists for
enantioselective synthesis (33).
In recent years, the cloning, sequencing, and expression of a variety
of Pseudomonas lipase genes have been reported (10, 15,
18, 22, 24, 30, 39). The genes can be divided into three homology
groups (assigned as classes I to III), where class III is only
distantly related to the other classes. Pseudomonas lipases
of classes I and II, including the broadly used lipases of
P. cepacia and P. glumae strains (class
II) as well as of P. aeruginosa strains (class I), need
a chaperone whose gene is located downstream of the lipase
gene for efficient secretion and folding of active lipase.
The deduced amino acid sequences of the chaperones belong to two
homology groups. For a detailed review of the biochemical and molecular
properties of Pseudomonas lipases, see references 12, 23, and 36.
The production of Pseudomonas lipases is currently carried
out with recombinant Pseudomonas strains harboring both the
lipase and its chaperone on a broad-host-range plasmid. With this
system, lipase production is increased 40- to 85-fold over that of the wild-type strain (16, 30). For recombinant P. cepacia ATCC 21808 (recently reclassified as Burkholderia
cepacia) harboring the large pMMB22-derived plasmid pHES12, only
moderate lipase productivity of 200 U/ml is obtained (15,
16). Moreover, lipase production by recombinant strains often
decreases over time, possibly because of the large size of the
plasmids. In addition, most Pseudomonas strains currently
used for lipase production are potential pathogens; thus, special
safety directions have to be considered. For these reasons and to
further increase lipase productivity, we aimed to develop a
heterologous E. coli expression system for the large-scale production of these important lipases.
Unfortunately, the expression of class I and II Pseudomonas
lipases in E. coli is hampered by the fact that a lipase
chaperone is necessary for effective folding of the lipase. To our
knowledge, high-level production of functional class I and II
Pseudomonas lipases in E. coli has not yet been achieved.
In this work, we report for the first time the overexpression of both
the lipase and its modified and truncated chaperone of P. cepacia ATCC 21808 in E. coli. In a simple and rapid in vitro refolding procedure, functionally active lipase can be obtained in large amounts and with a specific activity comparable to that of the
lipase purified from P. cepacia.
Materials.
Restriction enzymes, DNA-modifying enzymes, T4
DNA ligase, and Taq polymerase were from MBI Fermentas. The
Taq Dye Deoxy cycle sequencing kit was from Applied Biosystems. The DNA
gel extraction kit, Midi plasmid kit, Prep-spin plasmid kit, and
Ni-nitrilotriacetic acid (NTA) matrix were from Qiagen.
p-Nitrophenyl-palmitate (pNPP) was from Sigma. Peptone and
yeast extract were from Difco. All reagents were of analytical grade
unless otherwise stated.
Strains, plasmids, and media.
P. cepacia ATCC
21808 harboring plasmid pHES12 containing the lipase operon was kindly
provided by Boehringer Mannheim GmbH (15). E. coli BL321 (hsdS gal [
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
High-Level Formation of Active Pseudomonas cepacia
Lipase after Heterologous Expression of the Encoding Gene and Its
Modified Chaperone in Escherichia coli and Rapid In
Vitro Refolding
ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
PRL promoter. However, no
overexpression of the lipase chaperone was achieved in E. coli. Thus, chemical refolding of inactive lipase in the absence
of its chaperone yielded only 25 U/mg, compared to 3,470 U of the purified lipase secreted by recombinant P. cepacia per mg.
Sequence analysis of the chaperone revealed a high GC content (>90%)
in the 5' region of the gene and the presence of a putative membrane anchor at the N terminus. Hence, the 5' region of the gene was replaced
by a synthetic fragment, and the putative membrane anchor was removed
by deletion of the first 34 or 70 N-terminal amino acids. Only
truncation of the gene led to overexpression of the chaperone (up to
60%) in E. coli. With this chaperone, it was possible to
obtain for the first time in a simple refolding procedure a highly
active Pseudomonas lipase (classes I and II) expressed in
E. coli with a specific activity of up to 4,850 U/mg and a yield of 314,000 U/g of E. coli wet cells.
INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
cIts857
ind1 Dam7 nin5 lacUV5-T7 gene 1]) and E. coli DH5
(supE44
lacU169 [
80lacZM15] hsdR17 recA1 endA1
gyrA96 thi-1 relA1) were used for cloning and gene expression.
Gene expression.
Transformed E. coli BL321
(or E. coli DH5 for expression of truncated and
modified chaperone genes) with plasmids derived from
pCYTEXP1 containing the strong, temperature-inducible
PRL promoter was cultivated at 30°C until the optical
density at 578 nm was 0.8. Next, protein expression was induced by
shifting the cultivation temperature to 42°C. After 3 h of
induction, cells were harvested by centrifugation (10,000 × g, 10 min, 4°C).
Recombinant DNA techniques. Standard recombinant DNA methods were carried out as described by Sambrook et al. (32). The fluorescence-based dideoxy DNA cycle sequencing method was used for sequence determination. DNA sequencing was carried out with the Taq Dye Deoxy cycle sequencing kit and with a model 373A DNA sequencing system (Applied Biosystems) in accordance with the manufacturer's instructions. For PCR, DNA was amplified with the following cycle conditions: first step, 94°C for 4 min, 1 cycle; second step, 94°C for 1.5 min, 64°C for 2 min, and 72°C for 3 min, 25 cycles; and third step, 72°C for 4 min, with 8% dimethyl sulfoxide added to the PCR mixture.
Isolation of lipase inclusion bodies. E. coli cells from a 500-ml culture (2 g) were suspended in 25 ml of 50 mM Tris buffer (pH 8.0) containing 1 mM EDTA and disrupted by sonification with a Branson Sonifier W-250 (duty cycle, 35%; output control, 3; time, 7 min), and the pellet containing the insoluble inclusion bodies was washed several times with the same buffer. The inclusion bodies were dissolved in 20 ml of buffer B (10 mM Tris-HCl, 8 M urea, 0.1 M sodium phosphate [pH 8.0]) at room temperature for 1 h with mild stirring. Following centrifugation (10,000 × g, 15 min, 4°C), the solubilized and denatured lipase of the supernatant was subjected to refolding.
Purification of the lipase chaperone by Ni-NTA chromatography. E. coli cells from a 500-ml culture (2 g) were suspended in 20 ml of buffer A (10 mM Tris-HCl, 6 M guanidine-HCl, 0.1 M sodium phosphate [pH 8.0]) and lysed for 1 h at room temperature. Following centrifugation (10,000 × g, 15 min, 4°C), the supernatant was applied (0.2 ml/min) to an Ni-NTA column (1.6 by 5 cm) previously equilibrated with buffer. Unbound protein was eluted by washing the column with 10 volumes of buffer A, 5 volumes of buffer B, and 5 volumes of buffer C (10 mM Tris-HCl, 8 M urea, 0.1 M sodium phosphate [pH 6.3]). Finally, bound chaperone was eluted with buffer D (10 mM Tris-HCl, 8 M urea, 0.1 M sodium phosphate [pH 5.9]). The purity of the eluted chaperone was analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE).
Refolding of lipase in the presence of the chaperone. One hundred microliters of lipase solution obtained after inclusion body solubilization and containing 1 mg of denatured lipase was refolded in 100 ml of distilled water in the presence of different amounts of denatured (solubilized with 8 M urea) or native (not solubilized with urea) chaperone. After 24 h of refolding at 4°C, the lipase activity in the supernatant was determined with pNPP as the substrate.
In order to investigate the optimal ratio of lipase to chaperone, 0.1, 0.5, 1, and 3 mg (10 to 300 µl) of denatured and purified chaperones70HpHis and ompA70HpHis obtained after Ni-NTA chromatography were
used for the refolding of 1 mg of lipase as described above.
For simplified refolding, E. coli cells from a 500-ml
culture (2 g of cells) expressing a truncated chaperone
(70HpHis, ompA70HpHis, 34HpHis, or 34HpHis/ompA34HpHis)
were lysed in 20 ml of buffer B. After centrifugation (10,000 × g, 15 min, 4°C), the supernatant containing the denatured
(solubilized with 8 M urea) chaperone was used for in vitro refolding.
The amount of the chaperone in relation to the total protein in the
supernatant was estimated by SDS-PAGE analysis. Approximately 1 mg of
chaperone was used for the in vitro refolding of 1 mg of lipase as
described for the purified chaperone. In a similar way, native (not
solubilized with urea) chaperones (70HpHis, ompA70HpHis, and
34HpHis) were used for refolding. Instead of cell lysis in the
presence of urea, cells from a 500-ml culture were suspended in 25 ml
of 50 mM Tris buffer (pH 8.0) containing 1 mM EDTA and disrupted by
sonification with a Branson Sonifier W-250 (duty cycle, 35%; output
control, 3; time, 7 min). After centrifugation (10,000 × g, 15 min, 4°C), the supernatant containing the soluble
chaperone was used for refolding.
Analytical methods. Preparative gel electrophoresis was carried out with a 12.5% polyacrylamide gel as described by Laemmli (28), and proteins were stained with Coomassie brilliant blue R-250. The expression level was estimated as a percentage of the level of total cellular protein with Imagemaster VDS version 2.0 (Pharmacia).
For amino-terminal sequence analysis, purified proteins were subjected to SDS gel electrophoresis. After blotting, the polyvinylidene difluoride membrane was stained with Coomassie brilliant blue R-250, and the protein bands were cut out and used for amino-terminal sequence analysis. Amino-terminal sequence analysis was performed with a model 470A gas-phase sequencer (Applied Biosystems) in accordance with the manufacturer's instructions. Protein concentration was determined with the bicinchoninic acid protein assay kit by the enhanced method in accordance with the manufacturer's instructions (Pierce instructions 23220/23225) and with bovine serum albumin as the standard.Enzyme assay. The enzyme assay was performed with pNPP as the substrate by use of a Biochrom 4060 spectrophotometer (Pharmacia). Cleavage of pNPP was measured at 60°C with 0.1 M Tris buffer (pH 7.5) as described by Schmidt-Dannert et al. (35). One unit was defined as the amount of enzyme which caused the release of 1 µmol of p-nitrophenol per minute under the test conditions.
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RESULTS |
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Abstract
Introduction
Materials and methods
Results
Discussion
References
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Construction of plasmids. For the initial investigation of protein expression in E. coli, a 2,673-bp fragment containing the genes for the prelipase and the chaperone was cut from pHES12 with EcoRI and ligated into pCYTEXP1 linearized with the same enzyme, yielding pT-E(preLip-Hp) (Fig. 1).
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PRL promoter of pCYTEXP1, PCR was performed with pT-E(preLip-Hp) as a template and two oligonucleotides
introducing an NdeI site (including the ATG start codon) at
the 5' end of the prelipase gene and an EcoRI site at the 3'
end of the chaperone gene. After digestion of the resulting PCR
fragment (containing preLip-Hp) and pCYTEXP1 with
NdeI and EcoRI and subsequent ligation of the
products, expression vector pT-preLip-Hp was obtained (Fig. 1).
In two additional pCYTEXP1-derived expression plasmids containing
complete preLip-Hp, the original signal equence of the lipase gene was
removed, resulting in vector pT-Lip-Hp, and replaced by the
ompA signal sequence, yielding plasmid pT-ompALip-Hp.
pT-ompALip-Hp was obtained by amplifying the ompA
signal sequence of expression plasmid pT-ompABTL2
(34) with two oligonucleotides: the first complementary to
the 5' end of the ompA sequence and introducing an
NdeI site and the second complementary to the 3' end of the ompA sequence and to the 5' end of the mature lipase gene.
The PCR fragment thus obtained and an oligonucleotide complementary to
an SphI site inside the lipase gene served as primers for a second PCR with pT-preLip-Hp as a template. Digestion of both the
obtained PCR fragment and pT-preLip-Hp with NdeI and
SphI, followed by ligation, resulted in
pT-ompALip-Hp. pT-Lip-Hp was constructed by PCR with
pT-preLip-Hp as a template and with one primer complementary to
the 5' end of the mature lipase gene and introducing an NdeI
site (including the ATG start codon) and another primer complementary
to the 3' end of the chaperone gene and introducing an EcoRI
site. Digestion of both pT-preLip-Hp and the obtained PCR
fragment with EcoRI and NdeI, followed by
ligation, yielded pT-Lip-Hp (Fig. 1).
To fuse a six-histidine (His6) tag to the mature lipase
gene and the chaperone gene, both genes were subcloned in
plasmid pET20b(+) harboring a His6 tag (data not
shown). The tagged genes were transferred to pCYTEXP1 for
expression under the control of the PRL promoter by
amplification with oligonucleotides introducing NdeI
and EcoRI sites at the 5' and 3' ends, respectively. Cloning of these PCR fragments into pCYTEXP1 also linearized with
NdeI and EcoRI resulted in pT-HpHis and
pT-LipHis, respectively.
A modified chaperone gene (modHp) was constructed as
follows: the first 242 bp (ending at a SacII site) of the
gene were replaced by a codon-optimized (for E. coli) and
thus less GC-rich nucleotide sequence. To this end, four
oligonucleotides of 80 bp each and overlapping by approximately 15 bp
were synthesized and assembled by PCR. The terminal oligonucleotides
comprised an NdeI site (5' end) and a SacII site
(3' end inside the chaperone gene) for cloning of the PCR fragment
into pT-HpHis cut with the same enzymes, yielding pT-modHpHis
(Fig. 1).
5' truncation of the modified chaperone gene by 34 residues (102 bp)
and 70 residues (210 bp) resulted in two plasmids,
pT-34HpHis and pT-70HpHis, after PCR with two
primers: one introducing an NdeI site (including the ATG
start codon) at the new 5' end of the gene and another introducing an
EcoRI site at the 3' end of the gene. pT-modHpHis was
used as a template, and then the EcoRI- and
NdeI-digested PCR fragment was cloned into pT-modHpHis
cut with the same enzymes (Fig. 1).
Fusion of the ompA signal sequence to the modified and
truncated chaperone genes was carried out by PCR in a manner
similar to that described for pT-ompALip-Hp. The resulting
plasmids were named pT-ompA70HpHis and
pT-ompA34HpHis.
Overexpression of lipase in E. coli.
For initial
expression studies of recombinant lipase in E. coli, plasmid
pT-E(preLip-Hp) containing complete preLip-Hp
(EcoRI fragment) of pHES12 under the control of
PRL was constructed. However, after 3 h of
induction of transformed cells with this plasmid, no additional bands
of expressed lipase or chaperone were detected on Coomassie brilliant
blue R-250-stained SDS gels of cell extracts. A small band of 33 kDa
representing lipase was visualized after activity staining (data not
shown); this band corresponded to the low level of lipase activity (171 U/g of cells) measured after cell breakage (Table
1).
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PRL
promoter of pCYTEXP1, yielding pT-preLip-Hp and, after
deletion of the chaperone gene, pT-preLip. In addition, the
original signal sequence of the lipase gene was deleted in
pT-Lip-Hp and replaced by the ompA signal sequence, known to increase expression levels and to transport the protein across
the inner membrane, yielding pT-ompALip-Hp.
In contrast to pT-preLip-Hp, both vectors pT-Lip-Hp and
pT-ompALip-Hp (data not shown), in which the original
presequence is no longer present, led to overexpression of the lipase
(expression level, 40%) but not of the chaperone (Table 1). However,
the lipase was expressed as inclusion bodies, and most of them were inactive after purification; hence, no processing of the
ompA-lipase gene occurred. This conclusion was also
confirmed by amino-terminal sequencing. Only slightly increased levels
of expressed lipase activity were detected with
pT-preLip-Hp (293 U/g of cells) and pT-ompALip-Hp (547 U/g of cells), in which the lipase gene
is preceded by a signal sequence and the chaperone gene is present. The
purpose of the introduction of the ompA signal sequence in the construct pT-ompALip-Hp was to increase transport of
the lipase across the inner membrane and hence to increase lipase
activity due to the presence of a small amount of the chaperone in the periplasm. The product was internally retained; however, a small amount
of the lipase was exported to the periplasm. This result showed that
the lipase activity (547 U/g) of the construct with the
ompA signal sequence (pT-ompALip-Hp) was 13 times higher than that of the construct without ompA
(pT-Lip-Hp).
Overexpression of lipase chaperone in E. coli. Although the nucleotide sequence of the lipase gene has been elucidated (15), the nucleotide sequence of the chaperone gene located downstream of the lipase gene has not. Hence, an open reading frame of 1,032 bp, located 3 bp downstream of the lipase gene and encoding the lipase chaperone, was identified (Fig. 2). While the average GC content of the chaperone gene was 73%, a GC content of >90% was calculated for a 250-bp region at the 5' end. A putative Shine-Dalgarno sequence (GAAG) was found inside the 3' region of the lipase gene.
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PRL promoter, was constructed. However, no expression of the chaperone was
detected on SDS gels.
Both the GC-rich 250-bp 5' region of the chaperone gene and the
amino-terminal hydrophobic stretches of amino acids (residues 14 to 34 and residues 50 to 70), comprising a putative membrane anchor, might
adversely affect protein expression in E. coli. Thus, the
first 250 bp of the gene was replaced by a synthetic fragment,
thereby lowering the GC content from >90 to 60% and resulting in modHp. In addition, the putative membrane
anchor was removed by deleting the first 34 (34Hp) and 70 (70Hp) amino-terminal amino acids.
The modified and the truncated chaperone genes were inserted into
pCYTEXP1, yielding pT-modHpHis, pT-34HpHis,
and pT-70HpHis. Expression levels of 10% were observed for the
truncated chaperones 34HpHis (34 kDa) and 70HpHis (31 kDa)
(Fig. 3), whereas with modHp,
no additional band was observed after SDS-PAGE analysis of E. coli cells (data not shown). Fusion of the ompA signal
sequence to the truncated genes (pT-ompA34HpHis and
pT-ompA70HpHis) resulted in further sixfold and fourfold
increases in expression, respectively (Table 1 and Fig. 3). Although in
both ompA-fused genes the signal cleavage sites were
identical (Ala-Ala), cleavage of the ompA leader sequence
reached only 50% for ompA34Hp, as seen after SDS-PAGE
analysis (Fig. 3, lane 8) and as proved by amino-terminal sequencing.
Only ompA34Hp formed insoluble inclusion bodies in E. coli, while all other expressed chaperones were soluble.
Both 70HpHis and ompA70HpHis were purified by Ni-NTA
chromatography to >95% purity (Fig. 3).
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In vitro refolding of lipase expressed in E. coli. In a first attempt, denatured lipase isolated from inclusion bodies was subjected to different refolding procedures without the addition of the chaperone, including those described by Beer et al. (3) for the lipase of Rhizopus oryzae and by Frenken et al. (10) for the lipase of P. glumae (data not shown). However, only a poorly active lipase with a specific activity of 25 U/mg or less was obtained.
Denatured lipase isolated from inclusion bodies, however, was effectively refolded after 24 h of incubation at 4°C in distilled water in the presence of equal amounts (final concentrations, 5 to 10 µg/ml) of truncated chaperone. Refolding of 5 to 10 µg of purified and denatured mature lipase per ml with 5 to 30 µg of purified and denatured chaperone70HpHis or ompA70HpHis per ml
yielded a highly active lipase with a specific activity of 3,580 to
4,180 U/mg (Table 2), comparable to or
even slightly higher than the activity of the purified lipase secreted
from recombinant P. cepacia (3,470 U/mg) and purified
as described by Hom (15) and Kordel et al. (26).
Increasing the concentration of lipase 10-fold and, hence, also the
chaperone concentration in the refolding mixture decreased the
refolding efficiency significantly, by a factor of 8 (data not shown).
It was found that an excess of chaperone is needed for correct lipase
folding, as for other Pseudomonas lipase chaperones (1,
13, 20, 31). In those studies, it was found that at least one
lipase chaperone molecule was needed for the correct folding of one
lipase molecule, because the chaperone acts toward the lipase
noncatalytically.
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34HpHis, the lipase could be effectively refolded (Table 3). However, the most effective chaperone
for lipase refolding proved to be 70HpHis, yielding a highly active
lipase (up to 314,000 U of lipase per g of E. coli
cells) with the simplified refolding protocol, regardless of whether
the chaperone was used in a denatured (4,660 U/mg) or a native (4,850 U/mg) state. In contrast, 34HpHis and ompA70HpHis gave rise
to a more active lipase when they were used in a denatured state for
refolding.
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DISCUSSION |
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Abstract
Introduction
Materials and methods
Results
Discussion
References
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In previous work (15), the sequence of the lipase gene of P. cepacia ATCC 21808 was elucidated and was found to be highly homologous (>90%) to those of genes for other class I Pseudomonas lipases, genes including at least five cloned lipases from different P. cepacia strains (22, 24, 30). The sequence of the chaperone gene has not been determined. Sequence analysis revealed a high homology of the protein sequence (>90%) with those of chaperones from P. cepacia DSM 3959 (24) and M-12-33 (30) and Pseudomonas sp. strain KWI-56 (22), as expected.
The role of the chaperone gene located downstream of the lipase gene
for the in vivo and in vitro activation of Pseudomonas lipases has been well investigated for lipases of
Pseudomonas sp. strain 109 and P. aeruginosa
TE3285 (6, 31), belonging to class I, and lipases of
P. cepacia DSM 3959 (1, 13, 14, 24),
Pseudomonas sp. strain KWI-56 (21, 22), and
P. glumae PG1 (9, 11), belonging to class
II. Unlike the situation for other industrially relevant microbial
lipases, however, no efficient system for the production of large
amounts of active Pseudomonas lipase in a biologically safe
heterologous host such as E. coli has been described.
Since the overexpression of both the lipase and the chaperone needed
for in vitro activation is a prerequisite for the economic production
of lipase in E. coli, we subcloned the gene cluster for
the lipase and the chaperone into E. coli expression
vectors pUC19, pET20b(+) (data not shown), and pCYTEXP1.
Overexpression of the lipase gene (up to 40%) was achieved
only under the control of the strong, temperature-inducible PRL promoter and when the original signal sequence was
either removed or replaced by an ompA signal sequence known
to increase protein expression in E. coli
(4). Comparable high expression levels for
Pseudomonas lipases (classes I and II) in E. coli have been reported for the prelipase of
Pseudomonas sp. strain KWI-56 (29) and the mature
lipase of P. aeruginosa TE3285 (31). The formation of inactive and insoluble inclusion bodies has also been
reported for several Pseudomonas lipases (classes I and II) expressed at higher levels in E. coli (7, 29,
31). Low lipase activities (171 to 547 U/g of cells) were
detected in cell lysates of recombinant E. coli cells
harboring plasmids containing both the lipase gene and the chaperone
gene of P. cepacia; similar observations were made with
Pseudomonas lipases expressed in E. coli
(9, 11, 20, 22, 24, 31, 39).
Most Pseudomonas lipase chaperones are only poorly expressed
in E. coli, and expression had to be observed by a
highly sensitive method, such as Western blotting (1, 9, 11,
13, 14, 21). In keeping with these findings, we found no
overexpression of unmodified P. cepacia ATCC 21808 lipase chaperone in E. coli, although the gene was
placed under the control of the strong PRL promoter.
Sequence analysis identified a GC-rich (>90%) region at the 5' end of
the chaperone gene which might affect transcription in E. coli. However, even when we decreased the GC content to 60% by
replacement with a 250-bp synthetic, codon-optimized DNA fragment, we
could not increase expression. Frenken et al. (9, 11) showed
that the lipase chaperone of P. glumae expressed at low
levels in E. coli as well as in wild-type P. glumae is located in the periplasmic space and anchored to the
inner membrane by an amino-terminal peptide stretch. It is generally
assumed that lipase chaperones of Pseudomonas are involved
both in translocation and in folding of lipase during its secretion
(6, 9, 11, 20, 22, 24, 41). When we investigated the
amino-terminal region of the chaperone, we found two hydrophobic
stretches (residues 14 to 34 and residues 50 to 70) which might
function as a membrane anchor and in turn hamper protein overexpression
by blocking translocation in E. coli. Truncation of the
amino terminus by 34 and 70 residues, respectively, led to
overexpression of the chaperone in E. coli which could
be further increased (from 10% to 60%) by fusion of the truncated
genes to an ompA signal sequence.
In vitro refolding of Pseudomonas lipases (classes I and II)
expressed so far in E. coli resulted in poorly active
lipase preparations, with 5 to 10% of the activity of the native
enzyme (13, 19, 21, 33). In contrast, by using overexpressed truncated chaperones either in the native or in the denatured state, we
could, for the first time, quantitatively refold a
Pseudomonas lipase overexpressed in E. coli
(100% specific activity) with high yields (up to 314,000 U/g of
E. coli cells) in a simple refolding procedure. We
concluded that neither the truncated N-terminal part (70 residues) of
the chaperone nor the fused ompA signal sequence
(70HpHis) had any influence on the folding activity, while
ompA34HpHis (not processed) solubilized (with 8 M urea) from
inclusion bodies no longer showed any folding activity. Best refolding
results were obtained when the lipase and the chaperone were present in
similar amounts in the refolding mixture, as was observed for the in
vitro refolding of other lipases from Pseudomonas (1,
33). The fact that denatured chaperones 70HpHis,
ompA70HpHis, and 34HpHis could be applied in the refolding
suggested that the active conformation of denatured chaperones is very
quickly restored upon dilution in the refolding mixture, thus allowing a simple and economic refolding of denatured lipases by use of crude
chaperones overproduced in E. coli and solubilized with urea. With this procedure, as much as 314,000 U of lipase per g of wet
cells in a highly pure state and ready to be used in biotechnological
applications could be easily obtained. To further simplify the
production of active lipase, a coexpression system in which both the
lipase and the chaperone are located on one plasmid and each gene is
placed under the control of the strong PRL promoter is
being developed.
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ACKNOWLEDGMENTS |
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Dinh Thi Quyen gratefully acknowledges a scholarship from the Konrad Adenauer Foundation, Bonn, Germany. We thank Boehringer Mannheim for providing the recombinant P. cepacia strain.
We thank Volker Nödinger for N-terminal sequencing and H. Atomi for helpful discussions.
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FOOTNOTES |
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* Corresponding author. Mailing address: Institut für Technische Biochemie, Universität Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany. Phone: 49-711-685-3192. Fax: 49-711-685-4569. E-mail: rolf.d.schmid{at}rus.uni-stuttgart.de.
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REFERENCES |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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Applied and Environmental Microbiology, February 1999, p. 787-794, Vol. 65, No. 2
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