- Email: [email protected]
Construction of improved vectors for protein production in Pseudomonas aeruginosa
Gene, 172 (1996) 163-164 0 1996 Elsevier Science B.V. All rights reserved.
GENE
163
0378-l 119/96/$15.00
09624
Construction
of improved
vectors for protein production
in
Pseudomonas aeruginosa (Expression
vector; protein
production;
Alison A. Watson, Richard Centrefor
Molecular and Cellular Biology,
Received by P.A. Manning:
2 August
T7 promoter;
chloramphenicol
acetyl transferase;
T7 RNA polymerase)
A. Alm and John S. Mattick University of Queensland, Brisbane, Qld. 4072, Australia
1995; Revised/Accepted:
21 September/27
September
1995; Received at publishers:
15 January
1996
SUMMARY
We report the construction of two cloning vectors that are based on the Pseudomonas-Escherichia shuttle vector, pUCP19. The new vectors, pUCPKS and pUCPSK, contain a significantly expanded multiple cloning site (MCS) with an adjacent T7 promoter sequence. In conjunction with specifically engineered host strains encoding an inducible T7 RNA polymerase, these vectors allow the controlled production of plasmid-encoded proteins in both Escherichia coli and Pseudomonas aeruginosa to analyse the spectrum of products encoded by cloned segments of DNA. The usefulness of these vectors was demonstrated by expressing the chloramphenicol acetyltransferase (CAT)-encoding gene.
The nonenteric bacterium Pseudomonas aeruginosa (Pa) is a Grampathogen which possesses a large spectrum of biochemical pathways involved in a diverse range of activities. The expression of cloned genes is widely used to confirm the coding potential of cloned DNA segments. Limitations, however, are often encountered when attempting to express cloned Pa genes in heterologous hosts, such as Escherichia coli (EC), because of differences in codon usage (West and Iglewski, 1988) and considerstructure (Rothmel able variation of the Pa promoter et al., 1991). A T7 expression system for protein synthesis in Pa has been recently developed, whereby a T7 RNPencoding gene under lacUV5 control has been stably
integrated into the genome (Brunschwig and Darzins, 1992). However, the broad-host-range vectors used in this
PVUI
Correspondence to: J.S. Mattick, Centre for Molecular and Cellular Biology, University of Queensland, St. Lucia, Qld. 4072, Australia. Tel. (61-7) 3365-4446; Fax (61-7) 3365-4388; e-mail: [email protected] Abbreviations: A, absorbance (1 cm); bp, base pair(s); CAT, chloramphenicol acetyl transferase; cat, gene encoding CAT, IPTG, isopropylo-d-thiogalactopyranoside; kb, kilobase or 1000 bp; lacUV5, lac promoter carrying the UV5 mutation; MCS, multiple cloning site(s); nt, nucleotide(s); ori, origin of DNA replication; PAGE, polyacrylamidegel electrophoresis; RNP, RNA polymerase; SDS, sodium dodecyl sulfate; SF, stabilizing fragment. PZZ SO378-1119(96)00026-l
PVUI Fig. 1. The structure of pUCPKS. The shuttle plasmid pUCP19 and the general cloning vector pBluescriptI1 (KS+ or SK+) were digested with PuuI, and the 2.77-kb (containing the SF) and 1.9-kb (containing the T7 promoter and MCS) fragments purified, respectively. These fragments were ligated together using standard procedures, and a blue colony selected to ensure proper regeneration of the 1acZ coding sequence. The MCS was sequenced to ensure its integrity.
164 system are large (> 6.5 kb), and only offer a small number of unique
restriction
(Brunschwig
sites downstream
and Darzins,
of the T7 promoter
1992).
We have constructed two new vectors useful for T7-based protein synthesis in Pa. The vectors were constructed by ligating fragments from the parental vectors pUCP19 (Schweizer, 1991) and pBluescriptI1 SK+/KS+ (Fig. 1). The resulting vectors (pUCPSK and pUCPKS) contained the stabilizing fragment (SF) which allows maintenance of this plasmid in Pseudomonas species from
pUCP19, selection),
and the extended MCS (including blue/white the ColEl ori and the T7 promoter sequence
from pBluescript (Fig. 1). Tnese new vectors provide unique sites in the MCS for 22 different restriction enzymes
to allow greater manipulation of DNA fragments while also allowing direct analysis of gene expression in both EC and Pa without the need for additional
subcloning. The utility
of these vectors cat gene
promoterless
was tested by cloning
from
pCAT30
and
the
pCAT11
(Dobrowolski, 1991) downstream of the T7 promoter element between the BamHI and Hind111 sites of pUCPKS and pUCPSK, generating plasmids pAW144 and
A1234 kDa
PAW 145, respectively. All four plasmids were introduced and Pa ADD1976, which both into both EC JMlOg(DE3) contain chromosomal copies of the gene for T7 RNP under lacUV5 control. SDS-PAGE analysis of whole cell
-97.4
-66.0 -45.0
extracts from both EC and Pa after induction of the lac promoter demonstrated the production of increasing quantities of a 25-kDa protein in cells containing pAW144 or pAW145, which is consistent with the size of CAT (data not shown). Following specific labelling of any
-31.0 *
-21.5 -14.5
- 6.5
plasmid-encoded proteins with [35S]methionine, the 25-kDa CAT protein was visualised in cells containing
BI
2
either not
34
pAW144 the
or pAW145
parental
plasmids
(Fig. 2A, B; lanes 2, 4) but pUCPKS
or
pUCPSK
(Fig. 2A, B; lanes 1 and 3). kDa -97.4 -66.0
ACKNOWLEDGMENTS
-45.0 -31.0
This work was supported by grants from the Australian Research Council and the Australian National Health and Medical Research Council.
+ -21.5 -14.5
- 6.5 REFERENCES Fig. 2. T7-dependent
expression
of
cat. Cells containing
plasmids
pUCPKS (lane 1), pUCPSK (lane 3) pAW144 (lane 2) and pAW145 (lane 4) in Ec JM109(DE3) (A) or Pa ADD1976 (B) were grown to A 600nm--0 5, harvested, and resuspended in M9 medium plus 0.5% methionine assay medium (D&o). After a further 1 h growth, production of T7 RNA polymerase was induced with 1 mM IPTG for 30 min. Rifampicin (200 ug/ml) was added and incubation continued for 30-60 min. Plasmid-encoded proteins were pulse-labelled with [?S]methionine for 10 min, and samples were resuspended in 1 x sample buffer and analysed by 0.1% SDS/l2% PAGE and autoradiography. The specifically labelled 25-kDa CAT protein (arrowed) is seen exclusively in cells containing pAW144 or pAW145.
Brunschwig,
E. and Darzins
A.: A two-component
T7 system
for the
overexpression of genes in Pseudomonas ueruginosu. Gene 111 (1992) 35-41. Dobrowolski, P.: Plasmids with easily excisable cut gene cartridges. Gene 102 (1991) 1399140. Rothmel, R.K.. Chakrabarty. A.M., Berry, A. and Darzins, A.: Genetic systems in Pseudomonas. Meth. Enzymol. 204 (1991) 4855514. Schweizer, H.P.: Escl~erichia-Pselrdomonas shuttle vectors derived from pUC18!19. Gene 97 (1991) 1099112. West, S.E.H. and Tglewski, B.H.: Codon usage in Pseudomonas uerugiIZO.SCI. Nucleic Acids Res. 16 ( 1988) 9323 9334.
GENE
163
0378-l 119/96/$15.00
09624
Construction
of improved
vectors for protein production
in
Pseudomonas aeruginosa (Expression
vector; protein
production;
Alison A. Watson, Richard Centrefor
Molecular and Cellular Biology,
Received by P.A. Manning:
2 August
T7 promoter;
chloramphenicol
acetyl transferase;
T7 RNA polymerase)
A. Alm and John S. Mattick University of Queensland, Brisbane, Qld. 4072, Australia
1995; Revised/Accepted:
21 September/27
September
1995; Received at publishers:
15 January
1996
SUMMARY
We report the construction of two cloning vectors that are based on the Pseudomonas-Escherichia shuttle vector, pUCP19. The new vectors, pUCPKS and pUCPSK, contain a significantly expanded multiple cloning site (MCS) with an adjacent T7 promoter sequence. In conjunction with specifically engineered host strains encoding an inducible T7 RNA polymerase, these vectors allow the controlled production of plasmid-encoded proteins in both Escherichia coli and Pseudomonas aeruginosa to analyse the spectrum of products encoded by cloned segments of DNA. The usefulness of these vectors was demonstrated by expressing the chloramphenicol acetyltransferase (CAT)-encoding gene.
The nonenteric bacterium Pseudomonas aeruginosa (Pa) is a Grampathogen which possesses a large spectrum of biochemical pathways involved in a diverse range of activities. The expression of cloned genes is widely used to confirm the coding potential of cloned DNA segments. Limitations, however, are often encountered when attempting to express cloned Pa genes in heterologous hosts, such as Escherichia coli (EC), because of differences in codon usage (West and Iglewski, 1988) and considerstructure (Rothmel able variation of the Pa promoter et al., 1991). A T7 expression system for protein synthesis in Pa has been recently developed, whereby a T7 RNPencoding gene under lacUV5 control has been stably
integrated into the genome (Brunschwig and Darzins, 1992). However, the broad-host-range vectors used in this
PVUI
Correspondence to: J.S. Mattick, Centre for Molecular and Cellular Biology, University of Queensland, St. Lucia, Qld. 4072, Australia. Tel. (61-7) 3365-4446; Fax (61-7) 3365-4388; e-mail: [email protected] Abbreviations: A, absorbance (1 cm); bp, base pair(s); CAT, chloramphenicol acetyl transferase; cat, gene encoding CAT, IPTG, isopropylo-d-thiogalactopyranoside; kb, kilobase or 1000 bp; lacUV5, lac promoter carrying the UV5 mutation; MCS, multiple cloning site(s); nt, nucleotide(s); ori, origin of DNA replication; PAGE, polyacrylamidegel electrophoresis; RNP, RNA polymerase; SDS, sodium dodecyl sulfate; SF, stabilizing fragment. PZZ SO378-1119(96)00026-l
PVUI Fig. 1. The structure of pUCPKS. The shuttle plasmid pUCP19 and the general cloning vector pBluescriptI1 (KS+ or SK+) were digested with PuuI, and the 2.77-kb (containing the SF) and 1.9-kb (containing the T7 promoter and MCS) fragments purified, respectively. These fragments were ligated together using standard procedures, and a blue colony selected to ensure proper regeneration of the 1acZ coding sequence. The MCS was sequenced to ensure its integrity.
164 system are large (> 6.5 kb), and only offer a small number of unique
restriction
(Brunschwig
sites downstream
and Darzins,
of the T7 promoter
1992).
We have constructed two new vectors useful for T7-based protein synthesis in Pa. The vectors were constructed by ligating fragments from the parental vectors pUCP19 (Schweizer, 1991) and pBluescriptI1 SK+/KS+ (Fig. 1). The resulting vectors (pUCPSK and pUCPKS) contained the stabilizing fragment (SF) which allows maintenance of this plasmid in Pseudomonas species from
pUCP19, selection),
and the extended MCS (including blue/white the ColEl ori and the T7 promoter sequence
from pBluescript (Fig. 1). Tnese new vectors provide unique sites in the MCS for 22 different restriction enzymes
to allow greater manipulation of DNA fragments while also allowing direct analysis of gene expression in both EC and Pa without the need for additional
subcloning. The utility
of these vectors cat gene
promoterless
was tested by cloning
from
pCAT30
and
the
pCAT11
(Dobrowolski, 1991) downstream of the T7 promoter element between the BamHI and Hind111 sites of pUCPKS and pUCPSK, generating plasmids pAW144 and
A1234 kDa
PAW 145, respectively. All four plasmids were introduced and Pa ADD1976, which both into both EC JMlOg(DE3) contain chromosomal copies of the gene for T7 RNP under lacUV5 control. SDS-PAGE analysis of whole cell
-97.4
-66.0 -45.0
extracts from both EC and Pa after induction of the lac promoter demonstrated the production of increasing quantities of a 25-kDa protein in cells containing pAW144 or pAW145, which is consistent with the size of CAT (data not shown). Following specific labelling of any
-31.0 *
-21.5 -14.5
- 6.5
plasmid-encoded proteins with [35S]methionine, the 25-kDa CAT protein was visualised in cells containing
BI
2
either not
34
pAW144 the
or pAW145
parental
plasmids
(Fig. 2A, B; lanes 2, 4) but pUCPKS
or
pUCPSK
(Fig. 2A, B; lanes 1 and 3). kDa -97.4 -66.0
ACKNOWLEDGMENTS
-45.0 -31.0
This work was supported by grants from the Australian Research Council and the Australian National Health and Medical Research Council.
+ -21.5 -14.5
- 6.5 REFERENCES Fig. 2. T7-dependent
expression
of
cat. Cells containing
plasmids
pUCPKS (lane 1), pUCPSK (lane 3) pAW144 (lane 2) and pAW145 (lane 4) in Ec JM109(DE3) (A) or Pa ADD1976 (B) were grown to A 600nm--0 5, harvested, and resuspended in M9 medium plus 0.5% methionine assay medium (D&o). After a further 1 h growth, production of T7 RNA polymerase was induced with 1 mM IPTG for 30 min. Rifampicin (200 ug/ml) was added and incubation continued for 30-60 min. Plasmid-encoded proteins were pulse-labelled with [?S]methionine for 10 min, and samples were resuspended in 1 x sample buffer and analysed by 0.1% SDS/l2% PAGE and autoradiography. The specifically labelled 25-kDa CAT protein (arrowed) is seen exclusively in cells containing pAW144 or pAW145.
Brunschwig,
E. and Darzins
A.: A two-component
T7 system
for the
overexpression of genes in Pseudomonas ueruginosu. Gene 111 (1992) 35-41. Dobrowolski, P.: Plasmids with easily excisable cut gene cartridges. Gene 102 (1991) 1399140. Rothmel, R.K.. Chakrabarty. A.M., Berry, A. and Darzins, A.: Genetic systems in Pseudomonas. Meth. Enzymol. 204 (1991) 4855514. Schweizer, H.P.: Escl~erichia-Pselrdomonas shuttle vectors derived from pUC18!19. Gene 97 (1991) 1099112. West, S.E.H. and Tglewski, B.H.: Codon usage in Pseudomonas uerugiIZO.SCI. Nucleic Acids Res. 16 ( 1988) 9323 9334.