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Construction of an efficient overproducer clone of HinfI restriction endonuclease using the polymerase chain reaction
Oene. 88 (1990) i-5 Elsevier
!
GENE 03457
Construction of an efficient overproducer clone of Hinfl restriction endonuclease using the polymerase chain reaction (Recombinant DNA; Escherichia coli; overexpression; stem-loop structure; positive retroregulator; ribosome-binding site; plasmid)
Cynthia M. Skoglun&, Hamilton O. Smith b and Srinivasan Chandrasegaran" Departments of ° Environmental Health Sciences. School of Hygiene and Public Health. and b Molecular Biology and Genetics. School of Medicine. The Johns Hopkins University. Baltimore. MD 21205 (U.S.A.) Tel. (301)955-3650 Received by F. Barany: 11 October 1989 Accepted: 9 November 1989
SUMMARY
We describe the use of the polymerase chain reaction (PCR) technique to alter transcriptional and translational signals surrounding a gene so as to achieve overexpression in Escherichia cog. By changing the ribosome-binding site sequence preceding the hinflR gene to match the consensus E. cog signal and by adding a transcription terminator sequence immediately following the gene, the yield of Hinfl was increased about tenfold over that obtained from the natural Haemophilus influenzae signals. The addition of the positive retroregnlator stem-loop sequence derived from the cry~tal protein-encoding gene of Bacillus thuringiensis downstream from the hinflR gene further increased yields by twofold to a level of 13~o of the total cellular protein.
INTRODUCTION
We recently reported the cloning and sequencing of the Hint ENase and MTase genes, R and M, from H. influenzae Rf (Chandrasegaran et al., 1988). One clone described in that work carded the two genes on a 2.3-kb
Correspondence to: Dr. S. Chandrasegaran, Department of Environmental Health Sciences, School of Hygiene and Public Health, 615 N. Wolfe Street, The Johns Hopkins University, MD 21205 (U.S.A.) Tel. (301) 955-3651; Fax (301)955-0617. Abbreviations: A, absorbance; aa, amino acid(s); Ap, ampicillin;bp, base pair(s); BSA, bovine serum albumin; ENase, restriction endonuclease; EtdBr, ethidium bromide; H., Haemophilus;hinflR, gene encoding Hint ENasc; HPLC, high-performance liquid chromatography; IPTG, isopropyl-p.v.thiogalactopyranosidc; kb, kilobase(s) or 1000bp; nt, nucleotide(s); oligo, oligodeoxyribonucleotidc; PCR, polymerase chain reaction; R, restriction; R, resistance; RBS, ribosome-binding site; RRS, retroregulator sequence; SDS, sodium dodecyi sulfate; tac promoter, hybrid trp.lac promoter; 7', terminator; Tc, tetracycline; [ ], denotes plasmid-carrier state. 0378-1119/90/$03.50 © 1990ElsevierScience Publishers B.V.(BiomedicalDivision)
fragment inserted into the polylinker region of pUCI9 in reverse orientation to the lacUV5 promoter. This clone expressed the two genes from native H. influen:ae transcription and translation signals and yielded ENase amounting to no more than I ~ of total cellular protein. In subsequent work (unpublished) we obtained ENase yields of no better than 0.5% when the hinflR gene was located downstream from the Pt,c promoter in a pBR322 derivative (pSKI0) propagated in E. coll. Overproducing strains facilitate biochemical and physical studies of the enzymes, thus we sought ways to increase yields by altering translational signals for the ENase-encoding gnne. An optimal RBS for initiation of translation is generally a prerequisite for high-level synthesis of proteins. However, the rate of mRNA breakdown also plays an important role in determining the leve| of gene express~on (Brawerman, 1987). Wong and Chang (1986) reported that a 47-bp stemloop structure (,dO = -30.4 kcal) downstream from the crystal protein gene of B. thuringiensis, when placed in the region 3' to a gene, increases mRNA half-life by fivefold in E. cell and increases protein yields proportionately. Our
aim was to utilize both an optimized RBS and the positive retroregnlator stem-loop structure to obtain greatly increased yields of Hinfl. Furthermore, we have demonstrated the utility of the PCR technique (Saiki etal., 1988) for easy redesigning of translational signals.
mixtures were pooled and extracted once with 1:1 phenol/chloroform and twice with chloroform. The DNA was ethanol-precipitated and resuspended in 20/d TE buffer (10 mM Tris. HCI pH 7.5/1 mM EDTA). The DNA was then cleaved with BamHl to generate cohesive ends and gel-purified.
MATERIALS AND METHODS
(d) Cloning procedures and growth of cells
(a) Bacterial strains and plasmids Recombinant plasmids were transformed into E. coli RB791iq cells which carry the iaclq allele on the chromosome (Brunt and Ptashne, 1981). Plasmid pSK5 is a derivative of pACYCI84 carrying the hhallM gene on a 1.0-kb fragment inserted into the EcoRl site (Schoner et al., 1983). This plasmid expresses the M. Hhall MTase constitutively and was present in RB791 cells whenever the hinflR gone was introduced on a separate compatible plasmid. The M. Hhall MTase modifies Hinfl sites and provides protection against chromosomal cleavage. The vector pSK10 (Chandrasegaran et al., 1988) was derived by inserting the [acIq gene into the EcoRl site of pDR540 (Pharmacia). Plasmid pUCHinf2.3, carrying both the hinflR and hinflM genes, is described elsewhere (Chandrasegaran eta]., 1988). Plasmid pBBo, a modified pUC19 plasmid with a Bgill linker inserted at the Sacl site, was kindly provided by J.-F. Tomb.
(b) Enzymes, biochemicals and oligos Oligo primers for PCR and the positive retroregulator sequence were synthesized with an Applied Biosystem DNA Synthesizer using cyanoethyl phosphoramidite chemistry and purified by reversed-phase HPLC. Restriction enzymes and I"4 DNA ligase were from New England Biolabs and were used as recommended. IPTG was from Boehringer-Mannheim. PCR reagents were purchased as a GeneAmp Kit from Perkin-Elmer-Cetus.
(c) PCR reactions Plasmid pUCHinf2.3 DNA linearized with BamHI was used as template. PCR reactions (100~tl) contained 0.25 nmol of each primer/200 /~M of each ,tNTP/10 mM Tris. HCI pH 8.3 (at 25°C)/50 mM KCI/I.5 mM MgCI2/0.01 ~o (w/v) gelatin/l ng of template DNA/5 units of Taq DNA polymerase. Reaction mixtures (run in quadruplicate) were overlayed with mineral oil and reactions were carried out using the Perkin-Elmer-Cetus Thermal Cycler. Initial template denaturation was programmed for 1.5 min. Thereafter, the cycle profile was programmed as follows: 2 rain at 37°C (annealing), :5min at 72°C (extension), and I min at 94°C (denaturation). This profde was repeated for 25 cycles and the final 72°C extension step was increased to 7 min. The aqueous layers of the reaction
The PCR-generated DNA containing the hinflR gene was ligated into BamHI-cleaved pSKI0 or pBBo expression vectors and the DNA was transfected into RB791iq[pSK5] cells made competent as described by Chung and Miller (1988). After appropriate antibiotic selection (see next paragraph), several clones were picked and plasmid DNA was examined by restriction analysis for presence of the hinflR gone fragment in correct orientation to the vector promoters (Maniatis et al., 1982). To produce enzyme, plasmid-containing RB791iq cells were grown at 37 °C with shaking in 2 × concentrated TY medium (1.6~o tryptone/1 ~o yeast extract/0.5% NaCI pH 7.2) supplemented with 20 pg Tc/ml (except for those cells carrying the pUCHinf2.3 plasmid) and 50pg Ap/ml. IPTG was added to a concentration of I mM when the cell density reached Aeoo = 0.8. The cells were incubated overnight (12 h) with shaking.
(e) ENase assays Cells from a 5-ml sample of culture medium were harvested by centrifugation, resuspended in 0.5 ml sonication buffer (50 mM Tris. HCI pH 8/14 mM 2-mercaptoethanol), and disrupted by sonication (3 × 5 s each). The cellular debris was centrifuged and the supernatant was diluted tenfold in sonication buffer to restore the concentration to that of the original culture. Eight serial twofold dilutions with sonication buffer were then made to provide a rang~ of enzyme concentrations for assay. Reaction mixtures (I00/~I) contained I0 mM Tris. HCI pH 9/I0 mM MgCl2/7mM 2-mercaptoethanol/50~g of BSA per ml/10~g plasmid pTZI9R (U.S. Biochemicals)/10~l of diluted crude enzyme. Incubation was at 37 °C. To analyze the progress of each reaction, I0-/~I samples were removed from each of the dilution series reaction tubes at 5, I0, 15, 20, 30, and 40rain, l~I of dye solution (100raM EDTA/0.1 ~o bromphenol blue/0.1% xylene cyanol/50% glycerol) was added, arid the samples were electrophoresed on a 1~o agarose gel. Bands were stained with 0.5 #g EtdBr/ml and visualized with 310-nm ultraviolet light. The amount of activity was calculated from the greatest dilution of enzyme that produced a complete digestion in 10 min as determined by visual inspection. All activities were determined on the basis of at least two independent experiments. In general, activity measurements were reproducible within plus or minus 50~o. A unit of Hinfl activity is the amount
that produces a complete digestion of I pg ofplasmid DNA in I h at 37°C under standard buffer conditions.
RESULTS AND DISCUSSION
(a) Construction of a new RBS by PCR
To change the translational signals ofthe Hinfl restriction gene using PCR, we synthesized two oligo primers as shown in Fig. 1. The primer for the 5' end of the gene was a 43-nt oligo containing an optimal translation initiation site consisting of a strong RBS (GGAGGT) and an adjacent 7-bp spacer just preceding the ATG start codon (Chernak and Smith, 1989). Since two possible start codons for the hinflR gene have been proposed (Chandrasegaran et al., 1988), we designed the primer to anneal to the second start site to avoid a mixture of two forms of the enzyme. Also, we replaced the second and third codons with degenerate codons (AAT to AAC and GAT to GAC) to disrupt a potential secondary structure in the message and to improve codon usage. The 55-bp primer for the Y end of the gene anneals to the terminal six codons of the gene and is immediately followed by the tvpA transcription terminator (Christie et al., 1981). BamHl restriction sites are placed near the 5' ends of each primer to facilitate ligation of the amplified DNA into vectors. Through the PCR process, the additional non-
annealing sequences contained at the 5' ends of each primer become incorporated into the amplified product. This is the same strategy used by Scharfet al. (1986) to generate clones of the human ~-globin and HLA-DQA sequences in the bacteriophage M 13. (b) Construction of overprodueer clones using the PCRmodified hinflR gene The PCR-generat,~d hinflR gene fragment was ligated into the BamHl site oftwo vectors: pSKI0, possessing the strong Ptac promo:er and pBBo (Fig. 2) containing the lacUV5 promoter. Clones were obtained that carried the hinflR gene in the correct orientation for expression from the efficient vector promoters. These were examined for enzyme production. As can be seen in Figs. 3 and 4, these clones produced significantly greater amounts of H/nfl than clones carrying the natural transcription and/or translation signals. The Pta¢ pSKI0 clone gave an estimated ENase yield of 5 ~ of total cellular protein and 20 million units of activity per liter of culture. This compares to an upper estimate of 0.5% yield in the corresponding unmodified gene clone. It is apparent that the Pta~ promoter by itself is insufficient for high expression of the gene product; modifying the RBS sequence and adding a transcription pRRSHInfi H/nil pRRS
5' arlmer H/nell E=~l
BamHI 80 7.bp epaur 5' TA GGATCC GGAGGT TTAAAAT ~ Met ArT TCC GAC C lie 8er A|p
Ag~] G.4D GAA Ash Asp Glu
~,
__.
~,.
aAA~'rcAaA~maTAc~aaaaA~TCTAaAa~mAccmcAaacA~c~a~ BQIII ~ III Smal Xbal Pstl Xmal
3'
3' mimer T T A A C G T GC
CG GC GC GC CG
18-bp complement 3' TAT TAC CGA TAC T T r ~ T A A A A A A A A T lie Met Ale Met Lye
BamHI CCTAGG AT 5'
Fig. 1. Sequences of the 5' and 3' oligo primers used to introduce new translation and termination signals into the hinflR gene during PCR amplification. Both primers are flanked by BamHl sites. SD represents the Shine-Dalgnrno ~onsensus RBS for £. col~ and the 7-bp spacer separates the RBS from the ATG start ¢,odon. Start trod stop codons are shaded; open boxes enclose changes of T -, C in the second and third codons of the gene. The 18-bp complement sequence is complementary to the end of the hinflR gene.
Fig. 2. Structure of plasmids pBBo, pRRS, and pRRSH/nfl. The Saci site in the polylinkerofpUCI9 was replaced by a Bf/ll site to form pBBo. The retroregnlator sequence (RRS) shown in Fig. 5 was inserted into the Bglll site ofpBBo to form pRRS and ill©FCR-ir,odifi~d ;~TF, gc,~: web inserted at the upstre~n BamHl site ofpRRS to form pRRSH/nfl. Amp, ApR gene.
kDa
1
2
3
4
5
6
7
8
9
Vector Promoter RB8
hinflR
Transcdplion Positive Yield Terminator Retroregulator % protein
Activity
200 97.4
~
.... ~
~ ~,~ ~ i ~
pSK10
Ptac
TAAGG
m
~
0.5
<0.1
putt9 (2.3 kb)
uvs
TAAGG
m
m
1
S
pSKlO
Ptac
C~e~C-~T
-~--
--
5
20
pBBo
UV5
GGAGGT
---
~-
7
60
pBBo
UV5
GGAGGT
'-~
"--
8
60
........ 1 8 . 4
pEmo
uvs
GGAGST
".~
-~-
8
e0
......
pBBo
UV5
GGAGGT
--
+
13
100
o~ •
Hinfl
14.3
Fig. 3. Analysis of proteins from Hinfl overproducer strains by SDS-polyacrylamide gel electrophoresis. Cells from a 5-ml sample of culture were centrifuged, resuspended in 0.5 ml sonication buffer, sonicated 15 s, and the sonicate was clarified by centrifugation. Protein in a 20-pl sample of each crude extract was analyzed by electrophoresis on 0.1% SDS/12% polyacrylamide gels as described by Laemmli (1970). The host cell for all the plasmids is RB791iq and inductions were with ! mM IPTG for 12 h. Lanes: I and 2, pSK$ + pSK 10, no induction and induction, respectively; 3, pUCHlnf2.3, induced; 4, pSK5 plus pSKI0 car~ing the PCR-modified hinflR gene, induced; $ to 8, pSK5 plus the PCR-modificd hinflR gene on plasmid pBBo plus or minus the RRS or terminator (T) as follows (all are induced): 5, -RRS, -T; 6, -RRS, + T; 7, + RRS, + T; 8, + RRS, -1"; 9, protein sttmdards (sizes are in kDa).
Fig. 4. Yields of Hinfl protein and activity from various overproducer strains. All hinflR genes are oriented as shown in the top diagram except for the pUCH/nf2.3 clone which contains both the R and M genes expressed from natural signals and in reverse orientation to the lacUV5 promoter. Protein yields are given as % of total cellular protein as determined by laser densitometry. The amount of H/nil ENase, as a % of total cellular protein, was determined from Coomassie-blue-stained gels by laser densitometry using the LKB222-010 UltroScan XL. The instrument automatically integrates the area under peaks. The accuracy and reproducibility for large peaks is good, but for small peaks that are just above the background bands, only an upper level estimate is obtained. Thus the amount of protein in weak bands may be significantly overestimated. Activity is in millions of units per liter of culture (MATERIALS AND METHODS, section e). The designations of the seven plasmids (from top to bottom) are: pSKHinfl, pUCHinf2.3, pSKTHinfl, pBBoHinfl, pBBoTHinfl, pRRSTHinfl and pRRSHinfl.
terminator increases ENase production by at least tenfold. The pBBo lacUV5 promoter clone yielded 7Yo of the total cellular protein as ENase and activity was about 60 million units per liter of culture. Although the lacUV5 promoter is weaker th~n the Ptac promoter, the somewhat better yield is probably due to the high copy .umber of the pBBo plasmid. Alteration of the putative natural TAAGG RBS to GGAGGT l~robably accounts largely for the increased yields from the two clones since pBBo clones with and without the trpA transcription terminator sequence give nearly the same yield (8~o vs. 7~o, respectively).
,o A TA CA CG CG AT CG TA AT CG AT GC GC CG AT
(c) Influence of the positive retroregulator sequence
The 47-bp stem-loop positive retroregulator structure of Wong and Chang (1986) flanked by Bglll sites (Fig. 5) was synthesized and cloned into the BglII site of pBBo to yield pRRS. PCR-gcnerated hinflR gene products with and without the trpA transcriptional terminator were inserted into the -,~a.".:H! site (Figs. 2 and 4). Presence of 47-bp stem-loop structure downstream from the terminus of the ENase gene (no trpA terminator) increased ENase yield
Bglll
AT
~
Bglll
5' CTAGATCTAGAATACT,~TCTAGATCTAG 3' ' '~'bal
'
Xbal
Fig. 5. Sequence ofthe 69-bp synthetic oligo (RRS) containing the 47-bp stem-loop retroregulator structure from B. thu~ngiensis plus flanking restriction sites for use in cloning. The stem-loop structure acts as a transcriptional terminator. The downward arrow indicates the position at which the RNA transcript terminates as identified by Wong and Chang (1986).
from 7% to 13% and the Hinfl activity increased from 60 to 100 million units per liter ofculture (Fig. 4). The positive retroregulator is ineffective when preceded by the trpA transcription terminator. In this case the retroregnlator structure would not be present at the 3' end of the mRNA and could not, therefore, act as a barrier to ribonuclease action as proposed by Wong and Chang (1986).
(d) Conclusions (1) Many gene products can be produced in relatively large amounts in E. coil by the simple application of strong promoters. However, in some cases high levels of transcription edone are not sufficient. If a gene is derived from eukaryotic sources or from prokaryotes unrelated to E. coil, the translational (RBS) signals may be inefficient, or the RBS may be sequestered by secondary mRNA structure (Barany, 1988). In some cases, the mRNA may be unstable (Newbury et al., 1987), or the protein product may have a short half-life due to cellular proteases (Chemak and Smith, 1989). With the hinflR gene, high levels of transcription achieved by the strong Ptao promoter or by the high copy number obtained with the pUClg-derived pBBo plasmid were insufficient to give more than a 1~o yield of ENase. The two potential start sites for the R gene (Chandrasegaran et al., 1988) appear to be preceded by relatively weak E. coli RBSs. Replacement of the putative TAAGG natural RBS ofthe hinflR gone by a GGAGGT RB S spaced optimally from the second ATG start codon increased enzyme yield by five- to tenfold. Addition of the stable retroregulator stem-loop in the downstream region of the 8ene increased yield by approximately an additional twofold. It appears that the major factor contributing to the increased yields is improved ribosome binding, however, since we also altered the second and third codons of the 8ene (without changing its aa sequence), it is also possible that changes in mRNA folding may be involved. (2) PCR appears to be the method of choice for altering the gene expression signals. There is no requirement for strategically placed restriction sites or for subcloning of engineered subfra~nents. One simply synthesizes the appropriate oligo primers with the new sequences incorporated into the 5' overhanging ends. These new sequences are then added during the PCR reaction. By placing restriction sites at the extreme 5' ends of the primers, the PCR products are readily inserted into appropriate plasmid vectors. We were able to easily add a new RBS and a transcription terminator, but the 47-bp retroregulator sequence was too large to add conveniently, so it was inserted in a separate step. This step need not be repeated in the future because plasmid pBBo, containing the stem-loop
structure at one end of the polylinker region, can now be used routinely as a cloning vector. The methods we have described should be generally applicable.
ACKNOWLEDGEMENTS
This work was partially supported by American Cancer Society Grant No. NP 52282. S.C. has a Junior Faculty Research Award from the American Cancer Society and is supported by N.I.H. Grant No. GM 42140. H.O.S. is an American Cancer Society Research Professor. C.M.S. is supportcxi by the NIEI-IS Training Grant No. 5-T32-ESO7141.
REFERENCES Baruny, F.: Overproduction, purification and crystallization of Taql restriction endonuclease. Oene 65 (1988) 167-177. Brawerman, G.: Determinants of messenger RNA stability. Cell 48 (1987) 5-6. Brent, R. and Ptashne, M.: Mechanismof action ofthe lexA ~ne product. Prec. Natl. Acad. Sci. USA 78 (1981) 4204-4208. Chandrasegarun, S., Lunnen, K.D., Smith, H.O. and Wilson, G.G.: Cloningand sequencing the Hinfl restriction and modificationgenes. Gene 70 (1988) 387-392. Chernak, J.M. and Smith, H.O.: Use of synthetic ribosome binding site for overproduction of the $B protein cf insmion sequence I~. Nucleic Acids Res. 17 (1989) 1933-1951. Christie, G.E., Farnham, PJ. and Platt, T.: Synthetic sites for transcriptiun termination and a functional comparison with u'yptophan oi~ron termination sites in vitro. Prec. Natl. Acad. Sci. USA 78 (1981) 4180-4184. ChunK, C.T. and Miller, R.H.: A rapid and convenient method for the preparation and storage of competent bacterial cells. Nucleic Acids Res. 16 (1988) 3580-3580. Laemmli, U.K.: Cleavage of structural proteins during the assembly of the head of bacteriophage 'i"4. Nature 227 (1970) 680-685. Maniatis, T., Fritsch, E.F. and Sambrook, J.: Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982. Newbury, S.F., Smith, N.H., Robinson, E.C., Hiles, I.D. and Higgins, C.F.: Stabilization of translationally active mRNA by prokaryotic REP sequences. Cell 48 (1987) 297-310. Salki, P.K., Gelfand, D.H., Stoffel, S., Scharf, SJ., Higuchi, R., Horn, G.T., Mullis, K.B. and Erlich, H.A.: Primer-directed enzymatic amplificationof DNA with a thermostable DNA polymerase.Science 239 (1988) 487-491. Scharf, S., Horn, G.T. and Eflich, H.A.: Direct cloning and sequence analysis of enzymatically amplifiedgenomic sequences. Science 233 (1986) 1076-1078. Schoner, B., Kelly, S. and Smith, H.O.: The nucleotide sequence of the Hhall restriction and modification genes l~om Haemophi,~s haemolyticus. Oene 24 (1983) 227-236. Wong, H.C. and Chang, S.: Identification of the positive retroregulator that stabilizes mRNAs in bacteria. Proc. Natl. Acad. Sci. USA 83 (1986) 3233-3237.
!
GENE 03457
Construction of an efficient overproducer clone of Hinfl restriction endonuclease using the polymerase chain reaction (Recombinant DNA; Escherichia coli; overexpression; stem-loop structure; positive retroregulator; ribosome-binding site; plasmid)
Cynthia M. Skoglun&, Hamilton O. Smith b and Srinivasan Chandrasegaran" Departments of ° Environmental Health Sciences. School of Hygiene and Public Health. and b Molecular Biology and Genetics. School of Medicine. The Johns Hopkins University. Baltimore. MD 21205 (U.S.A.) Tel. (301)955-3650 Received by F. Barany: 11 October 1989 Accepted: 9 November 1989
SUMMARY
We describe the use of the polymerase chain reaction (PCR) technique to alter transcriptional and translational signals surrounding a gene so as to achieve overexpression in Escherichia cog. By changing the ribosome-binding site sequence preceding the hinflR gene to match the consensus E. cog signal and by adding a transcription terminator sequence immediately following the gene, the yield of Hinfl was increased about tenfold over that obtained from the natural Haemophilus influenzae signals. The addition of the positive retroregnlator stem-loop sequence derived from the cry~tal protein-encoding gene of Bacillus thuringiensis downstream from the hinflR gene further increased yields by twofold to a level of 13~o of the total cellular protein.
INTRODUCTION
We recently reported the cloning and sequencing of the Hint ENase and MTase genes, R and M, from H. influenzae Rf (Chandrasegaran et al., 1988). One clone described in that work carded the two genes on a 2.3-kb
Correspondence to: Dr. S. Chandrasegaran, Department of Environmental Health Sciences, School of Hygiene and Public Health, 615 N. Wolfe Street, The Johns Hopkins University, MD 21205 (U.S.A.) Tel. (301) 955-3651; Fax (301)955-0617. Abbreviations: A, absorbance; aa, amino acid(s); Ap, ampicillin;bp, base pair(s); BSA, bovine serum albumin; ENase, restriction endonuclease; EtdBr, ethidium bromide; H., Haemophilus;hinflR, gene encoding Hint ENasc; HPLC, high-performance liquid chromatography; IPTG, isopropyl-p.v.thiogalactopyranosidc; kb, kilobase(s) or 1000bp; nt, nucleotide(s); oligo, oligodeoxyribonucleotidc; PCR, polymerase chain reaction; R, restriction; R, resistance; RBS, ribosome-binding site; RRS, retroregulator sequence; SDS, sodium dodecyi sulfate; tac promoter, hybrid trp.lac promoter; 7', terminator; Tc, tetracycline; [ ], denotes plasmid-carrier state. 0378-1119/90/$03.50 © 1990ElsevierScience Publishers B.V.(BiomedicalDivision)
fragment inserted into the polylinker region of pUCI9 in reverse orientation to the lacUV5 promoter. This clone expressed the two genes from native H. influen:ae transcription and translation signals and yielded ENase amounting to no more than I ~ of total cellular protein. In subsequent work (unpublished) we obtained ENase yields of no better than 0.5% when the hinflR gene was located downstream from the Pt,c promoter in a pBR322 derivative (pSKI0) propagated in E. coll. Overproducing strains facilitate biochemical and physical studies of the enzymes, thus we sought ways to increase yields by altering translational signals for the ENase-encoding gnne. An optimal RBS for initiation of translation is generally a prerequisite for high-level synthesis of proteins. However, the rate of mRNA breakdown also plays an important role in determining the leve| of gene express~on (Brawerman, 1987). Wong and Chang (1986) reported that a 47-bp stemloop structure (,dO = -30.4 kcal) downstream from the crystal protein gene of B. thuringiensis, when placed in the region 3' to a gene, increases mRNA half-life by fivefold in E. cell and increases protein yields proportionately. Our
aim was to utilize both an optimized RBS and the positive retroregnlator stem-loop structure to obtain greatly increased yields of Hinfl. Furthermore, we have demonstrated the utility of the PCR technique (Saiki etal., 1988) for easy redesigning of translational signals.
mixtures were pooled and extracted once with 1:1 phenol/chloroform and twice with chloroform. The DNA was ethanol-precipitated and resuspended in 20/d TE buffer (10 mM Tris. HCI pH 7.5/1 mM EDTA). The DNA was then cleaved with BamHl to generate cohesive ends and gel-purified.
MATERIALS AND METHODS
(d) Cloning procedures and growth of cells
(a) Bacterial strains and plasmids Recombinant plasmids were transformed into E. coli RB791iq cells which carry the iaclq allele on the chromosome (Brunt and Ptashne, 1981). Plasmid pSK5 is a derivative of pACYCI84 carrying the hhallM gene on a 1.0-kb fragment inserted into the EcoRl site (Schoner et al., 1983). This plasmid expresses the M. Hhall MTase constitutively and was present in RB791 cells whenever the hinflR gone was introduced on a separate compatible plasmid. The M. Hhall MTase modifies Hinfl sites and provides protection against chromosomal cleavage. The vector pSK10 (Chandrasegaran et al., 1988) was derived by inserting the [acIq gene into the EcoRl site of pDR540 (Pharmacia). Plasmid pUCHinf2.3, carrying both the hinflR and hinflM genes, is described elsewhere (Chandrasegaran eta]., 1988). Plasmid pBBo, a modified pUC19 plasmid with a Bgill linker inserted at the Sacl site, was kindly provided by J.-F. Tomb.
(b) Enzymes, biochemicals and oligos Oligo primers for PCR and the positive retroregulator sequence were synthesized with an Applied Biosystem DNA Synthesizer using cyanoethyl phosphoramidite chemistry and purified by reversed-phase HPLC. Restriction enzymes and I"4 DNA ligase were from New England Biolabs and were used as recommended. IPTG was from Boehringer-Mannheim. PCR reagents were purchased as a GeneAmp Kit from Perkin-Elmer-Cetus.
(c) PCR reactions Plasmid pUCHinf2.3 DNA linearized with BamHI was used as template. PCR reactions (100~tl) contained 0.25 nmol of each primer/200 /~M of each ,tNTP/10 mM Tris. HCI pH 8.3 (at 25°C)/50 mM KCI/I.5 mM MgCI2/0.01 ~o (w/v) gelatin/l ng of template DNA/5 units of Taq DNA polymerase. Reaction mixtures (run in quadruplicate) were overlayed with mineral oil and reactions were carried out using the Perkin-Elmer-Cetus Thermal Cycler. Initial template denaturation was programmed for 1.5 min. Thereafter, the cycle profile was programmed as follows: 2 rain at 37°C (annealing), :5min at 72°C (extension), and I min at 94°C (denaturation). This profde was repeated for 25 cycles and the final 72°C extension step was increased to 7 min. The aqueous layers of the reaction
The PCR-generated DNA containing the hinflR gene was ligated into BamHI-cleaved pSKI0 or pBBo expression vectors and the DNA was transfected into RB791iq[pSK5] cells made competent as described by Chung and Miller (1988). After appropriate antibiotic selection (see next paragraph), several clones were picked and plasmid DNA was examined by restriction analysis for presence of the hinflR gone fragment in correct orientation to the vector promoters (Maniatis et al., 1982). To produce enzyme, plasmid-containing RB791iq cells were grown at 37 °C with shaking in 2 × concentrated TY medium (1.6~o tryptone/1 ~o yeast extract/0.5% NaCI pH 7.2) supplemented with 20 pg Tc/ml (except for those cells carrying the pUCHinf2.3 plasmid) and 50pg Ap/ml. IPTG was added to a concentration of I mM when the cell density reached Aeoo = 0.8. The cells were incubated overnight (12 h) with shaking.
(e) ENase assays Cells from a 5-ml sample of culture medium were harvested by centrifugation, resuspended in 0.5 ml sonication buffer (50 mM Tris. HCI pH 8/14 mM 2-mercaptoethanol), and disrupted by sonication (3 × 5 s each). The cellular debris was centrifuged and the supernatant was diluted tenfold in sonication buffer to restore the concentration to that of the original culture. Eight serial twofold dilutions with sonication buffer were then made to provide a rang~ of enzyme concentrations for assay. Reaction mixtures (I00/~I) contained I0 mM Tris. HCI pH 9/I0 mM MgCl2/7mM 2-mercaptoethanol/50~g of BSA per ml/10~g plasmid pTZI9R (U.S. Biochemicals)/10~l of diluted crude enzyme. Incubation was at 37 °C. To analyze the progress of each reaction, I0-/~I samples were removed from each of the dilution series reaction tubes at 5, I0, 15, 20, 30, and 40rain, l~I of dye solution (100raM EDTA/0.1 ~o bromphenol blue/0.1% xylene cyanol/50% glycerol) was added, arid the samples were electrophoresed on a 1~o agarose gel. Bands were stained with 0.5 #g EtdBr/ml and visualized with 310-nm ultraviolet light. The amount of activity was calculated from the greatest dilution of enzyme that produced a complete digestion in 10 min as determined by visual inspection. All activities were determined on the basis of at least two independent experiments. In general, activity measurements were reproducible within plus or minus 50~o. A unit of Hinfl activity is the amount
that produces a complete digestion of I pg ofplasmid DNA in I h at 37°C under standard buffer conditions.
RESULTS AND DISCUSSION
(a) Construction of a new RBS by PCR
To change the translational signals ofthe Hinfl restriction gene using PCR, we synthesized two oligo primers as shown in Fig. 1. The primer for the 5' end of the gene was a 43-nt oligo containing an optimal translation initiation site consisting of a strong RBS (GGAGGT) and an adjacent 7-bp spacer just preceding the ATG start codon (Chernak and Smith, 1989). Since two possible start codons for the hinflR gene have been proposed (Chandrasegaran et al., 1988), we designed the primer to anneal to the second start site to avoid a mixture of two forms of the enzyme. Also, we replaced the second and third codons with degenerate codons (AAT to AAC and GAT to GAC) to disrupt a potential secondary structure in the message and to improve codon usage. The 55-bp primer for the Y end of the gene anneals to the terminal six codons of the gene and is immediately followed by the tvpA transcription terminator (Christie et al., 1981). BamHl restriction sites are placed near the 5' ends of each primer to facilitate ligation of the amplified DNA into vectors. Through the PCR process, the additional non-
annealing sequences contained at the 5' ends of each primer become incorporated into the amplified product. This is the same strategy used by Scharfet al. (1986) to generate clones of the human ~-globin and HLA-DQA sequences in the bacteriophage M 13. (b) Construction of overprodueer clones using the PCRmodified hinflR gene The PCR-generat,~d hinflR gene fragment was ligated into the BamHl site oftwo vectors: pSKI0, possessing the strong Ptac promo:er and pBBo (Fig. 2) containing the lacUV5 promoter. Clones were obtained that carried the hinflR gene in the correct orientation for expression from the efficient vector promoters. These were examined for enzyme production. As can be seen in Figs. 3 and 4, these clones produced significantly greater amounts of H/nfl than clones carrying the natural transcription and/or translation signals. The Pta¢ pSKI0 clone gave an estimated ENase yield of 5 ~ of total cellular protein and 20 million units of activity per liter of culture. This compares to an upper estimate of 0.5% yield in the corresponding unmodified gene clone. It is apparent that the Pta~ promoter by itself is insufficient for high expression of the gene product; modifying the RBS sequence and adding a transcription pRRSHInfi H/nil pRRS
5' arlmer H/nell E=~l
BamHI 80 7.bp epaur 5' TA GGATCC GGAGGT TTAAAAT ~ Met ArT TCC GAC C lie 8er A|p
Ag~] G.4D GAA Ash Asp Glu
~,
__.
~,.
aAA~'rcAaA~maTAc~aaaaA~TCTAaAa~mAccmcAaacA~c~a~ BQIII ~ III Smal Xbal Pstl Xmal
3'
3' mimer T T A A C G T GC
CG GC GC GC CG
18-bp complement 3' TAT TAC CGA TAC T T r ~ T A A A A A A A A T lie Met Ale Met Lye
BamHI CCTAGG AT 5'
Fig. 1. Sequences of the 5' and 3' oligo primers used to introduce new translation and termination signals into the hinflR gene during PCR amplification. Both primers are flanked by BamHl sites. SD represents the Shine-Dalgnrno ~onsensus RBS for £. col~ and the 7-bp spacer separates the RBS from the ATG start ¢,odon. Start trod stop codons are shaded; open boxes enclose changes of T -, C in the second and third codons of the gene. The 18-bp complement sequence is complementary to the end of the hinflR gene.
Fig. 2. Structure of plasmids pBBo, pRRS, and pRRSH/nfl. The Saci site in the polylinkerofpUCI9 was replaced by a Bf/ll site to form pBBo. The retroregnlator sequence (RRS) shown in Fig. 5 was inserted into the Bglll site ofpBBo to form pRRS and ill©FCR-ir,odifi~d ;~TF, gc,~: web inserted at the upstre~n BamHl site ofpRRS to form pRRSH/nfl. Amp, ApR gene.
kDa
1
2
3
4
5
6
7
8
9
Vector Promoter RB8
hinflR
Transcdplion Positive Yield Terminator Retroregulator % protein
Activity
200 97.4
~
.... ~
~ ~,~ ~ i ~
pSK10
Ptac
TAAGG
m
~
0.5
<0.1
putt9 (2.3 kb)
uvs
TAAGG
m
m
1
S
pSKlO
Ptac
C~e~C-~T
-~--
--
5
20
pBBo
UV5
GGAGGT
---
~-
7
60
pBBo
UV5
GGAGGT
'-~
"--
8
60
........ 1 8 . 4
pEmo
uvs
GGAGST
".~
-~-
8
e0
......
pBBo
UV5
GGAGGT
--
+
13
100
o~ •
Hinfl
14.3
Fig. 3. Analysis of proteins from Hinfl overproducer strains by SDS-polyacrylamide gel electrophoresis. Cells from a 5-ml sample of culture were centrifuged, resuspended in 0.5 ml sonication buffer, sonicated 15 s, and the sonicate was clarified by centrifugation. Protein in a 20-pl sample of each crude extract was analyzed by electrophoresis on 0.1% SDS/12% polyacrylamide gels as described by Laemmli (1970). The host cell for all the plasmids is RB791iq and inductions were with ! mM IPTG for 12 h. Lanes: I and 2, pSK$ + pSK 10, no induction and induction, respectively; 3, pUCHlnf2.3, induced; 4, pSK5 plus pSKI0 car~ing the PCR-modified hinflR gene, induced; $ to 8, pSK5 plus the PCR-modificd hinflR gene on plasmid pBBo plus or minus the RRS or terminator (T) as follows (all are induced): 5, -RRS, -T; 6, -RRS, + T; 7, + RRS, + T; 8, + RRS, -1"; 9, protein sttmdards (sizes are in kDa).
Fig. 4. Yields of Hinfl protein and activity from various overproducer strains. All hinflR genes are oriented as shown in the top diagram except for the pUCH/nf2.3 clone which contains both the R and M genes expressed from natural signals and in reverse orientation to the lacUV5 promoter. Protein yields are given as % of total cellular protein as determined by laser densitometry. The amount of H/nil ENase, as a % of total cellular protein, was determined from Coomassie-blue-stained gels by laser densitometry using the LKB222-010 UltroScan XL. The instrument automatically integrates the area under peaks. The accuracy and reproducibility for large peaks is good, but for small peaks that are just above the background bands, only an upper level estimate is obtained. Thus the amount of protein in weak bands may be significantly overestimated. Activity is in millions of units per liter of culture (MATERIALS AND METHODS, section e). The designations of the seven plasmids (from top to bottom) are: pSKHinfl, pUCHinf2.3, pSKTHinfl, pBBoHinfl, pBBoTHinfl, pRRSTHinfl and pRRSHinfl.
terminator increases ENase production by at least tenfold. The pBBo lacUV5 promoter clone yielded 7Yo of the total cellular protein as ENase and activity was about 60 million units per liter of culture. Although the lacUV5 promoter is weaker th~n the Ptac promoter, the somewhat better yield is probably due to the high copy .umber of the pBBo plasmid. Alteration of the putative natural TAAGG RBS to GGAGGT l~robably accounts largely for the increased yields from the two clones since pBBo clones with and without the trpA transcription terminator sequence give nearly the same yield (8~o vs. 7~o, respectively).
,o A TA CA CG CG AT CG TA AT CG AT GC GC CG AT
(c) Influence of the positive retroregulator sequence
The 47-bp stem-loop positive retroregulator structure of Wong and Chang (1986) flanked by Bglll sites (Fig. 5) was synthesized and cloned into the BglII site of pBBo to yield pRRS. PCR-gcnerated hinflR gene products with and without the trpA transcriptional terminator were inserted into the -,~a.".:H! site (Figs. 2 and 4). Presence of 47-bp stem-loop structure downstream from the terminus of the ENase gene (no trpA terminator) increased ENase yield
Bglll
AT
~
Bglll
5' CTAGATCTAGAATACT,~TCTAGATCTAG 3' ' '~'bal
'
Xbal
Fig. 5. Sequence ofthe 69-bp synthetic oligo (RRS) containing the 47-bp stem-loop retroregulator structure from B. thu~ngiensis plus flanking restriction sites for use in cloning. The stem-loop structure acts as a transcriptional terminator. The downward arrow indicates the position at which the RNA transcript terminates as identified by Wong and Chang (1986).
from 7% to 13% and the Hinfl activity increased from 60 to 100 million units per liter ofculture (Fig. 4). The positive retroregulator is ineffective when preceded by the trpA transcription terminator. In this case the retroregnlator structure would not be present at the 3' end of the mRNA and could not, therefore, act as a barrier to ribonuclease action as proposed by Wong and Chang (1986).
(d) Conclusions (1) Many gene products can be produced in relatively large amounts in E. coil by the simple application of strong promoters. However, in some cases high levels of transcription edone are not sufficient. If a gene is derived from eukaryotic sources or from prokaryotes unrelated to E. coil, the translational (RBS) signals may be inefficient, or the RBS may be sequestered by secondary mRNA structure (Barany, 1988). In some cases, the mRNA may be unstable (Newbury et al., 1987), or the protein product may have a short half-life due to cellular proteases (Chemak and Smith, 1989). With the hinflR gene, high levels of transcription achieved by the strong Ptao promoter or by the high copy number obtained with the pUClg-derived pBBo plasmid were insufficient to give more than a 1~o yield of ENase. The two potential start sites for the R gene (Chandrasegaran et al., 1988) appear to be preceded by relatively weak E. coli RBSs. Replacement of the putative TAAGG natural RBS ofthe hinflR gone by a GGAGGT RB S spaced optimally from the second ATG start codon increased enzyme yield by five- to tenfold. Addition of the stable retroregulator stem-loop in the downstream region of the 8ene increased yield by approximately an additional twofold. It appears that the major factor contributing to the increased yields is improved ribosome binding, however, since we also altered the second and third codons of the 8ene (without changing its aa sequence), it is also possible that changes in mRNA folding may be involved. (2) PCR appears to be the method of choice for altering the gene expression signals. There is no requirement for strategically placed restriction sites or for subcloning of engineered subfra~nents. One simply synthesizes the appropriate oligo primers with the new sequences incorporated into the 5' overhanging ends. These new sequences are then added during the PCR reaction. By placing restriction sites at the extreme 5' ends of the primers, the PCR products are readily inserted into appropriate plasmid vectors. We were able to easily add a new RBS and a transcription terminator, but the 47-bp retroregulator sequence was too large to add conveniently, so it was inserted in a separate step. This step need not be repeated in the future because plasmid pBBo, containing the stem-loop
structure at one end of the polylinker region, can now be used routinely as a cloning vector. The methods we have described should be generally applicable.
ACKNOWLEDGEMENTS
This work was partially supported by American Cancer Society Grant No. NP 52282. S.C. has a Junior Faculty Research Award from the American Cancer Society and is supported by N.I.H. Grant No. GM 42140. H.O.S. is an American Cancer Society Research Professor. C.M.S. is supportcxi by the NIEI-IS Training Grant No. 5-T32-ESO7141.
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