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Oligonucleotide-directed mutagenesis and subsequent expression of the corresponding recombinant proteins without changing the bacterial vector system
PHARMACEUTICA ACTA HELVETIAE ELSEVIER
Pharmaceutics Acta Helvetiae 72 (1997) 139-143
Oligonucleotide-directed mutagenesis and subsequent expression of the corresponding recombinant proteins without changing the bacterial vector system Martina Michael, Susanna Gerber, Jiirgen Fetzer, Gerd Folkers Department
of Pharmacy, ETH Zurich, Winterthurerstr.
*
190, 8057 Zurich, Switzerland
Received 16 July 1996; accepted 31 July 1996
Abstract A new bacterial vector was constructed that combines the attractive features of gene fusion vectors and phagemids. A gene of interest cloned into this new vector can either be expressed as fusion protein or be prepared as single-stranded template DNA within the same
system. Thus, time consuming subcloning procedures changing the bacterial vector according to the required method are avoided. As an example sequencing, expression and subsequent purification of site-directed mutants of herpes simplex virus type 1 thymidine kinase are discussed. Keywords:
Bacterial vector; Fusion protein; Single-stranded DNA; Site-directed mutagenesis
1. Introduction Suitable plasmid cloning vectors are available for nearly every problem arising in recombinant DNA technology. For example there are vectors constructed for easy identification of recombinant clones, for easy preparation of single-stranded copies in quantities sufficient for sequencing and for the enhanced expression of foreign genes. But if the combination of these possibilities is required, the change of the vector system will be recommended. Thus time consuming subcloning procedures are necessary. Expression vectors can be divided into three types: cytoplasmatic expression vectors, secretion vectors and gene fusion vectors. The ability to express cloned genes as fusion proteins containing a partner of known size and biological function can greatly simplify the isolation and purification of the gene product. With the commercially available pGEX system the protein of interest is expressed as cytoplasmatic N-terminal fusion with glutathione Stransferase (GST) from Schistoma japonicum and can be
* Corresponding author. Tel.: +41-l-2576060; fax: +41-l-2621580.
purified under non-denaturating conditions by affinity chromatography on immobilised glutathione. Protease recognition sites for thrombin or factor Xa are introduced to allow cleavage of the desired protein from the fusion product. The pGEX vectors contain the tat promotor and an internal lad4 gene for inducible, high-level and hostindependent expression. Phagemid vectors are plasmids that carry the origin of replication from the genome of a single-stranded filamentous bacteriophage. Segments of foreign DNA cloned into these vectors can be propagated as plasmids in the conventional way providing the stability and high yields of double-stranded DNA. When cells harboring these vectors are infected with a suitable filamentous bacteriophage the mode of replication changes, generating copies of one strand of the plasmid DNA. Such single-stranded DNAs are the templates of choice for DNA sequencing by the dideoxy chain-termination method, for site-directed mutagenesis using synthetic oligonucleotides and for generating DNA probes for hybridization with radiolabel in only one strand. In the present paper the construction of a new bacterial vector is described that allows the preparation of single-
0031-6865/97/$17.00 Copyright 0 1997 Elsevier Science B.V. All rights reserved. PII SO03 1-6865(97)00006-X
M. Michael et al. / Pharmaceuticu
140
stranded DNA as well as the expression of cloned genes as fusion proteins. We used this vector for site-directed mutagenesis of the gene coding for herpes simplex virus type 1 thymidine kinase (HSV 1 TK), for subsequent verification by dideoxy sequencing and finally for the expression of the corresponding recombinant proteins.
Acta Heluetiae 72 (1997) 139-143
with fresh LB-medium containing 50 pg/ml ampicillin and superinfected with M13K07 (4 X 10’ pfu/ml). After 1 h, kanamycin was added to a final concentration of 75 pg/ml. After 14-18 h of growing, single-stranded DNA was prepared from the supernatant according to standard methods (Sambrook et al., 1989). 2.5. Sequencing
2. Material
and methods
2.1. Materials E. coli strain 71/ 18 was used as the host for cloning procedures. The TK-deficient E. coli strain KY895, a gift from W.C. Summers (Liu and Summers, 1988) was used as the host for the expression of the GST-TK fusion protein. The plasmid pBR322-TK containing the gene for HSV 1 TK was a gift from S. McKnight. The plasmids pUCl8 and pBluescript II KS were purchased from Clontech and pGEX2T from Pharmacia. Enzymes used in molecular cloning were obtained from Boehringer. Chemicals were purchased from Sigma. 2.2. Construction
of the vector pGEX2T-fl
The origin of replication of the filamentous phage fl (fl ori) was cut out by SspI-PuuI restriction of the vector pBluescript II KS. The fragment was blunt-end ligated into the Asp1 cleaved vector pGEX2T yielding the phagemid vector pGEX2T-fl *. In order to find out the orientation of the inserted fl ori the vector was cleaved with NaeI. 2.3. Construction
of the uector pGEX2T-TKfl
Direct subcloning of the gene for HSV 1 TK into the pGEX-fl f phagemid construct was not possible. Synthetic DNA linkers coding for the amino acids upstream from the MluI recognition site in the open reading frame of tk followed by codons for MluI and SmaI recognition site and codons for the amino acids downstream from the SmaI site to the stop codon of the tk were cloned into the BamHI-EcoRI cleaved plasmid pUC18. The MluI-SmaI fragment of tk was inserted into this recombinant plasmid yielding pUC18-TK. Cleavage of pUClS-TK with EcoRI and BamHI generates a fragment containing the whole open reading frame of HSV 1 TK ready to subclone in frame into the multiple cloning site of pGEX2T-fl ’ yielding pGEX2T-TKf 1 * . 2.4. Preparation
of single-stranded
plasmid DNA
E. coli strain 7 1/ 18 was transformed with pGEX2T-f 1’ or pGEX2T-TKfl+. An overnight culture was diluted 1: 100
Dideoxy sequencing was carried out according to Sanger et al. (1977). The 19mer 5’-GTG GTG GCG ACC ATC CTC C-3’ was used as sequencing primer for all recombinant pGEX plasmids. In all successful mutations the full sequence of the tk gene was checked to ensure that no unwanted changes have occurred. 2.6. Site-directed
mutagenesis
Oligonucleotide-directed mutagenesis was carried out according to the phosphorothioate-based method described by Eckstein and co-workers (Taylor et al., 1985) using the kit from Amersham. We confined ourselves to summarizing the mutagenesis reaction below. More detailed information is provided by the kit protocol. The mutagenic oligonucleotide is annealed to this single-stranded template and extended by Klenow polymerase in the presence of T4 DNA ligase to generate a mutant heteroduplex. Singlestranded template molecules which remain unconverted into heteroduplex molecules during the priming and extension reaction are removed by T5 exonuclease digestion because otherwise they can lead to large reductions in mutagenic efficiency. Selective removal of a non-mutant strand is made possible by the incorporation of a thionucleotide into the mutant strand during in vitro synthesis. Eckstein observed that certain restriction enzymes cannot cleave phosphorothioate DNA. As a result, single-strand nicks are generated in DNA containing one phosphorothioate and one non-phosphorothioate strand. Such nicks present sites for exonuclease III, which can then be used to digest away all or part of the non-mutant strand of the cloned target sequence. The mutant strand is then used as a template to reconstruct the double-stranded closed circular molecule, thus creating a homoduplex mutant molecule. The 32mer 5’-GAG GAC AGA CAC ATC GCC CGC CTG GCC AAA CG-3’ and 5’-GGT GCG GTA TCT GCA AGG CGG CGG GTC GTG GC-3’ were used as the mutagenic oligonucleotides for the exchanges of Asp 215 of the HSV 1 TK into an Arg and for the exchange of Cys 251 into a Gly, respectively. The mutagenesis procedure can be analysed by taking samples after each step of the mutagenesis reaction as directed in the protocol of the kit. The samples were separated by electrophoresis at 80 V on
M. Michael et al. /Pharmaceutics Acta Heluetiae 72 Cl9971 139-143
a 0.6% agarose gel using 1 X TAE and 0.3 pg/ml ium bromide.
141
HSVl TK
ethiddCY&
2.7. Expression
and purification
of HSV I TK
E. coli strain KY895 harboring the pGEX2T-TKfl + vector were grown overnight at 37°C in LB medium containing ampicillin. The culture was diluted 1:lO with fresh medium and grown for 2 h at 25°C. Gene expression was induced by the addition of IPTG (isopropyl p-D-thiogalactopyranoside) to a final concentration of 100 FM. After 12- 18 h at 25°C bacteria were harvested by centrifugation, frozen, thawed, resuspended in lysis buffer (50 mM Tris pH 7.5, 10% glycerol, 5 mM EDTA, 1% Triton X-100,0.1 mM PMSF, 1 mM DTT, 150 pg/ml lysozyme) and lysed on ice by mild sonication. Centrifugation yielded the soluble part of total cell constituent. The GST-TK fusion protein was purified by glutathione affinity chromatography (150 mg glutathione agarose/2 ml bed volume) and cleaved with thrombin (3 NIH units/2 ml 20 mM Tris pH 8.4, 150 mM NaCl, 2.5 mM CaCl,, 0.1% Triton X-100, 1 mM DTT) directly on the column. (The column was regenerated by washing with 5 mM glutathione in 1 M Tris pH 8.4 in order to elute the GST part.) The flow through was pre-incubated with 50 PM thymidine for 12 h at 4°C and subsequently applied to an ATP affinity column (125 mg ATP agarose C-8 attachment/2 ml bed volume). The column was equilibrated with 10 mM MOPS (3-[N-Morpholino]propanesulfonic acid) pH 7.5, 1 mM EDTA, 1 mM DTT (DL-dithiothreitol), 50 PM thymidine, 0.1% Triton X-100. For elution this buffer with an additional 10 mM ATP was used. Expression and purification of the HSV 1 TK was monitored by SDS-PAGE. SDS-polyacrylamide electrophoresis was carried out either in horizontal gels using the PhastSystem from Pharmacia (with PhastGel Gradient lo-15 and PhastGel SDS buffer strips), or in vertical gels as described by Laemmli (1970).
3. Results and discussion A bacterial vector was constructed that enables both, the preparation of a single-stranded DNA template of the gene of interest and the expression of the corresponding protein as fusion protein. The constructed plasmid pGEX2T-fl contained the intergenic region of the filamentous bacteriophage fl (Fig. 1). According to the orientation of the inserted fl fragment superinfection of bacteria harboring this plasmid with the helper virus M13K07 resulted in the production of single-stranded sense or antisense copies of the plasmid DNA. Additionally the constructed
fl * ori
fl ori
480Q2I IDI/
pBR322 ori Fig. I. Construction of the vectors pGEX2T-fl* and pCEX2T-TKfl +. The origin of replication of the filamentous phage fl (fl ori) was blunt-end cloned into the Asp1 site of the pGEX2T vector yielding the pCEX2T-fl’ vector. In compliance with the open reading frame the gene encoding the HSV 1 TK was cloned into the multiple cloning site of pGEX2T-fl + yielding pGEX2T-TKfl ’. Additionally the vector contains the coding regions for the glutathione binding domain of the glutathione S-transferase (GST) and the thrombin recognition site (amino acid sequence LVPRGS) under control of the chimeric tat promotor (p,,,).
vector pGEX2T-fl contained the coding region for the glutathione binding domain of the glutathione S-transferase (GST) under control of the chimeric tat promotor. The coding sequence for a thrombin recognition site and a polycloning site were adjacent. Thus, a protein of interest cloned in frame into this polycloning site is expressed as N-terminal fusion with GST. Glutathione affinity chromatography and subsequent cleavage with thrombin provides the unfused recombinant protein. First we tested this new phagemid on its capabilities to produce template DNA and to overexpress GST. Preparation of single-stranded DNA and subsequent dideoxy sequencing according to standard methods (Sambrook et al., 1989) was carried out successfully. An oligonucleotide that annealed to the single-stranded template DNA derived from pGEX2T-fl+ just upstream the polycloning site was used as a sequencing primer for all recombinant pGEX vectors. As shown by SDS-PAGE the addition of IPTG to a culture of E. coli 7 I / 18 harboring pGEX2T-fl + resulted in the expression of large quantities of a protein with a molecular weight of 28 kDa which is in agreement with GST (Fig. 2). Thus, the new vector behaves as desired. Then we used this new phagemid for site-directed mutagenesis, sequencing and expression of Herpes Simplex Virus type 1 thymidine kinase (HSV 1 TK). For this
M. Michael et al./Phatmaceutica
142 1
2
3
kDa
M
-
66
-
4.5
-
31
-
21
-
14
Fig. 2. Expression of the GST part of the fusion protein. Gene expression was induced by the addition of IPTG to a bacterial culture of E. coli harboring the phagemid pCEX2T-fl + and monitored by SDS-PAGE. Whole protein of bacteria from 0.66 ~1 of non-induced (lane 1) and induced (lane 2) culture as well as 0.66 ~1 of the supematant (lane 3) containing the soluble protein constituent part of the cells were taken. Samples were solubilised in 2.5% SDS and boiled in the presence of 2-mercaptoethanol for 5 min before electrophoresis on a SDS-polyacrylamide gel using the PhastSystem from Pharmacia. Proteins were visualised by Coomassie Blue staining. The sizes &Da) of the molecular weight markers (lane M) are indicated.
purpose the wildtype tk gene was cloned in frame into the polycloning site of pGEX2T-fl + yielding pGEX2T-TKfl + as shown in Fig. 1. Preparation of single-stranded DNA and subsequent dideoxy sequencing of the wildtype tk gene using the above mentioned pGEX specific primer was carried out successfully. Additionally, both mutagenic oligonucleotides synthesized for site-directed mutagenesis were used as sequencing primers in order to check their correct annealing. Both annealed correctly and dideoxy sequencing was carried out without any problems. As the first step in oligonucleotide-directed mutagenesis is an annealing, priming and extension reaction analogous to dideoxy sequencing, we expected site-directed mutagenesis with our newly constructed phagemid being successful, despite the fact that most single-stranded based mutagenesis protocols recommended M 13 vectors. However, first attempts with our phagemid system resulted in extremely low mutagenic efficiency. The mutagenesis reaction was therefore analysed step by step. A sample was taken at each stage of the mutagenesis procedure and separated by electrophoresis on an agarose gel. The bands on the agarose gel can be assigned to the different forms of DNA produced during the mutagenesis procedure. After the first step most of the template DNA has been converted to a mixture of relaxed RF DNA and nicked DNA indicating that the mutant heteroduplex was generated
Acta Heluetiae 72 (1997) 139-143
successfully. However, the second step in the mutagenesis reaction, which is the complete removal of remaining single-stranded wildtype material, was not sufficient, resulting in a considerably high non-mutant background. This could be due to a minor quality of our single-stranded template DNA, which means that there is too much of co-purified single-stranded DNA of the helper phage. Hence the capacity limit of the T5 exonuclease digestion was reached. These findings confirm that our newly constructed phagemid system is suitable for oligonucleotidedirected mutagenesis but suggest some minor adjustments of the mutagenesis reaction protocol in order to improve the mutagenic efficiency. Expression and purification of the wildtype and the mutated proteins were monitored by SDS-PAGE as shown in Fig. 3. Cytoplasmatic expression of foreign proteins in E. coli often results in the formation of insoluble aggregates. Smith and Johnson (1988) described that most of different GST fusion proteins tested by them were completely or at least partly soluble. However, overexpression of GST-TK fusion protein at 37°C results in inclusion bodies. Attempts to dissolve this aggregates in denaturating agents and refold the protein result in completely inactive preparations. But reduction of the growth tempera-
Ml234567
Fig. 3. SDS-PAGE analysis of expression and purification of HSV 1 TK wildtype and mutants. E. coli harboring the phagemid pGEX2T-TKfl + were grown and harvested as described in Section 2. Whole protein of bacteria from 200 ~1 of induced culture was taken (lane 1). 150 pg of protein from the soluble supematant (lane 2) and 20 pg of glutathione affinity purified fusion protein (lane 3). 20 pg of the purified fusion protein were incubated with 0.02 units thrombin for 3 h (lane 41, for 6 h (lane 5) and for 24 h (lane 6). The completely digested TK was further purified by ATP affinity chromatography (lane 7, 5 kg). Samples were solubilised in 2% SDS and boiled in the presence of 2-mercaptoethanol for 5 min before electrophoresis on a vertical 10% SDS-polyacrylamide gel. Proteins were visualised by Coomassie Blue staining. The sizes &Da) of the molecular weight markers (lane M) are indicated.
M. Michael et al./ Phamaceutica
ture from 37 to 25°C solves this problem, yielding partly soluble GST-TK fusion protein (Fetzer and Folkers, 1992). The fusion proteins can be cleaved by digestion with site-specific proteases, such as thrombin or factor Xa. But often this cleavage is inefficient or non-specific (Dixon et al., 1993). Factor Xa is most frequently the restriction protease of choice, because it cleaves at the C-terminal end of its recognition site resulting in cleavage products with the original first amino acid at the N-terminal end. However, we decided on thrombin because its unmatched stability when dissolved, despite its disadvantage that the cleavage products start always with two additional amino acids Gly-Ser at the N-terminus. We prefer digesting the fusion protein while being bound to the glutathione affinity column. Thus, TK appeared in the eluent while the GST part remained bound to the column. However, thrombin cleavage of the GST-TK fusion protein was surprisingly not site-specific. Thrombin cleaved at two additional and hitherto unknown sites resulting in a truncated TK missing the first 33 amino acids (Fetzer et al., 1994). For final purification ATP affinity chromatography has to be carried out in order to separate the truncated HSV 1 TK from thrombin and the small peptides derived from the non-
Acta Heluetiae 72 (1997) 139-143
143
specific stepwise thrombin cleavage. This two-step purification procedure resulted in homogeneously pure and enzymatically active wildtype HSV 1 TK (Fetzer et al., 1994). Both mutants of HSV 1 TK, namely D215R and C25 1G, could be purified by this procedure as well. Quantities of l-2 mg recombinant enzyme per liter of bacterial culture were yielded.
References Dixon, J.E., Hakes, D. and Guan, K.L. (1993) In: Methods in Molecular Genetics, Vol. 2. Academic Press. Fetzer, J. and Folkers, G. (1992) Pharm. Pharmacol. Lett. 2, 112-l 14. Fetzer, J., Michael, M., Bohner, T., Hofbauer, R. and Folkers, G. (1994) Prot. Exp. Purif. 5, 432-441. Laemmli, U.K. (1970) Nature 227, 680-685. Liu, Q. and Summers, W.C. (1988) Virology 163, 638-642. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: a Laboratory Manual, 2nd Ed. CSH Press. Sanger, F., Nicklen, S. and Coulson, A.R. (1977) PNAS (USA) 74, 5463-5467. Smith, D.B. and Johnson, K.S. (1988) Gene 67, 31-40. Taylor, J.W., Ott, J. and Eckstein, F. (1985) Nucleic Acids Res. 13(24), 8765-8785.
Pharmaceutics Acta Helvetiae 72 (1997) 139-143
Oligonucleotide-directed mutagenesis and subsequent expression of the corresponding recombinant proteins without changing the bacterial vector system Martina Michael, Susanna Gerber, Jiirgen Fetzer, Gerd Folkers Department
of Pharmacy, ETH Zurich, Winterthurerstr.
*
190, 8057 Zurich, Switzerland
Received 16 July 1996; accepted 31 July 1996
Abstract A new bacterial vector was constructed that combines the attractive features of gene fusion vectors and phagemids. A gene of interest cloned into this new vector can either be expressed as fusion protein or be prepared as single-stranded template DNA within the same
system. Thus, time consuming subcloning procedures changing the bacterial vector according to the required method are avoided. As an example sequencing, expression and subsequent purification of site-directed mutants of herpes simplex virus type 1 thymidine kinase are discussed. Keywords:
Bacterial vector; Fusion protein; Single-stranded DNA; Site-directed mutagenesis
1. Introduction Suitable plasmid cloning vectors are available for nearly every problem arising in recombinant DNA technology. For example there are vectors constructed for easy identification of recombinant clones, for easy preparation of single-stranded copies in quantities sufficient for sequencing and for the enhanced expression of foreign genes. But if the combination of these possibilities is required, the change of the vector system will be recommended. Thus time consuming subcloning procedures are necessary. Expression vectors can be divided into three types: cytoplasmatic expression vectors, secretion vectors and gene fusion vectors. The ability to express cloned genes as fusion proteins containing a partner of known size and biological function can greatly simplify the isolation and purification of the gene product. With the commercially available pGEX system the protein of interest is expressed as cytoplasmatic N-terminal fusion with glutathione Stransferase (GST) from Schistoma japonicum and can be
* Corresponding author. Tel.: +41-l-2576060; fax: +41-l-2621580.
purified under non-denaturating conditions by affinity chromatography on immobilised glutathione. Protease recognition sites for thrombin or factor Xa are introduced to allow cleavage of the desired protein from the fusion product. The pGEX vectors contain the tat promotor and an internal lad4 gene for inducible, high-level and hostindependent expression. Phagemid vectors are plasmids that carry the origin of replication from the genome of a single-stranded filamentous bacteriophage. Segments of foreign DNA cloned into these vectors can be propagated as plasmids in the conventional way providing the stability and high yields of double-stranded DNA. When cells harboring these vectors are infected with a suitable filamentous bacteriophage the mode of replication changes, generating copies of one strand of the plasmid DNA. Such single-stranded DNAs are the templates of choice for DNA sequencing by the dideoxy chain-termination method, for site-directed mutagenesis using synthetic oligonucleotides and for generating DNA probes for hybridization with radiolabel in only one strand. In the present paper the construction of a new bacterial vector is described that allows the preparation of single-
0031-6865/97/$17.00 Copyright 0 1997 Elsevier Science B.V. All rights reserved. PII SO03 1-6865(97)00006-X
M. Michael et al. / Pharmaceuticu
140
stranded DNA as well as the expression of cloned genes as fusion proteins. We used this vector for site-directed mutagenesis of the gene coding for herpes simplex virus type 1 thymidine kinase (HSV 1 TK), for subsequent verification by dideoxy sequencing and finally for the expression of the corresponding recombinant proteins.
Acta Heluetiae 72 (1997) 139-143
with fresh LB-medium containing 50 pg/ml ampicillin and superinfected with M13K07 (4 X 10’ pfu/ml). After 1 h, kanamycin was added to a final concentration of 75 pg/ml. After 14-18 h of growing, single-stranded DNA was prepared from the supernatant according to standard methods (Sambrook et al., 1989). 2.5. Sequencing
2. Material
and methods
2.1. Materials E. coli strain 71/ 18 was used as the host for cloning procedures. The TK-deficient E. coli strain KY895, a gift from W.C. Summers (Liu and Summers, 1988) was used as the host for the expression of the GST-TK fusion protein. The plasmid pBR322-TK containing the gene for HSV 1 TK was a gift from S. McKnight. The plasmids pUCl8 and pBluescript II KS were purchased from Clontech and pGEX2T from Pharmacia. Enzymes used in molecular cloning were obtained from Boehringer. Chemicals were purchased from Sigma. 2.2. Construction
of the vector pGEX2T-fl
The origin of replication of the filamentous phage fl (fl ori) was cut out by SspI-PuuI restriction of the vector pBluescript II KS. The fragment was blunt-end ligated into the Asp1 cleaved vector pGEX2T yielding the phagemid vector pGEX2T-fl *. In order to find out the orientation of the inserted fl ori the vector was cleaved with NaeI. 2.3. Construction
of the uector pGEX2T-TKfl
Direct subcloning of the gene for HSV 1 TK into the pGEX-fl f phagemid construct was not possible. Synthetic DNA linkers coding for the amino acids upstream from the MluI recognition site in the open reading frame of tk followed by codons for MluI and SmaI recognition site and codons for the amino acids downstream from the SmaI site to the stop codon of the tk were cloned into the BamHI-EcoRI cleaved plasmid pUC18. The MluI-SmaI fragment of tk was inserted into this recombinant plasmid yielding pUC18-TK. Cleavage of pUClS-TK with EcoRI and BamHI generates a fragment containing the whole open reading frame of HSV 1 TK ready to subclone in frame into the multiple cloning site of pGEX2T-fl ’ yielding pGEX2T-TKf 1 * . 2.4. Preparation
of single-stranded
plasmid DNA
E. coli strain 7 1/ 18 was transformed with pGEX2T-f 1’ or pGEX2T-TKfl+. An overnight culture was diluted 1: 100
Dideoxy sequencing was carried out according to Sanger et al. (1977). The 19mer 5’-GTG GTG GCG ACC ATC CTC C-3’ was used as sequencing primer for all recombinant pGEX plasmids. In all successful mutations the full sequence of the tk gene was checked to ensure that no unwanted changes have occurred. 2.6. Site-directed
mutagenesis
Oligonucleotide-directed mutagenesis was carried out according to the phosphorothioate-based method described by Eckstein and co-workers (Taylor et al., 1985) using the kit from Amersham. We confined ourselves to summarizing the mutagenesis reaction below. More detailed information is provided by the kit protocol. The mutagenic oligonucleotide is annealed to this single-stranded template and extended by Klenow polymerase in the presence of T4 DNA ligase to generate a mutant heteroduplex. Singlestranded template molecules which remain unconverted into heteroduplex molecules during the priming and extension reaction are removed by T5 exonuclease digestion because otherwise they can lead to large reductions in mutagenic efficiency. Selective removal of a non-mutant strand is made possible by the incorporation of a thionucleotide into the mutant strand during in vitro synthesis. Eckstein observed that certain restriction enzymes cannot cleave phosphorothioate DNA. As a result, single-strand nicks are generated in DNA containing one phosphorothioate and one non-phosphorothioate strand. Such nicks present sites for exonuclease III, which can then be used to digest away all or part of the non-mutant strand of the cloned target sequence. The mutant strand is then used as a template to reconstruct the double-stranded closed circular molecule, thus creating a homoduplex mutant molecule. The 32mer 5’-GAG GAC AGA CAC ATC GCC CGC CTG GCC AAA CG-3’ and 5’-GGT GCG GTA TCT GCA AGG CGG CGG GTC GTG GC-3’ were used as the mutagenic oligonucleotides for the exchanges of Asp 215 of the HSV 1 TK into an Arg and for the exchange of Cys 251 into a Gly, respectively. The mutagenesis procedure can be analysed by taking samples after each step of the mutagenesis reaction as directed in the protocol of the kit. The samples were separated by electrophoresis at 80 V on
M. Michael et al. /Pharmaceutics Acta Heluetiae 72 Cl9971 139-143
a 0.6% agarose gel using 1 X TAE and 0.3 pg/ml ium bromide.
141
HSVl TK
ethiddCY&
2.7. Expression
and purification
of HSV I TK
E. coli strain KY895 harboring the pGEX2T-TKfl + vector were grown overnight at 37°C in LB medium containing ampicillin. The culture was diluted 1:lO with fresh medium and grown for 2 h at 25°C. Gene expression was induced by the addition of IPTG (isopropyl p-D-thiogalactopyranoside) to a final concentration of 100 FM. After 12- 18 h at 25°C bacteria were harvested by centrifugation, frozen, thawed, resuspended in lysis buffer (50 mM Tris pH 7.5, 10% glycerol, 5 mM EDTA, 1% Triton X-100,0.1 mM PMSF, 1 mM DTT, 150 pg/ml lysozyme) and lysed on ice by mild sonication. Centrifugation yielded the soluble part of total cell constituent. The GST-TK fusion protein was purified by glutathione affinity chromatography (150 mg glutathione agarose/2 ml bed volume) and cleaved with thrombin (3 NIH units/2 ml 20 mM Tris pH 8.4, 150 mM NaCl, 2.5 mM CaCl,, 0.1% Triton X-100, 1 mM DTT) directly on the column. (The column was regenerated by washing with 5 mM glutathione in 1 M Tris pH 8.4 in order to elute the GST part.) The flow through was pre-incubated with 50 PM thymidine for 12 h at 4°C and subsequently applied to an ATP affinity column (125 mg ATP agarose C-8 attachment/2 ml bed volume). The column was equilibrated with 10 mM MOPS (3-[N-Morpholino]propanesulfonic acid) pH 7.5, 1 mM EDTA, 1 mM DTT (DL-dithiothreitol), 50 PM thymidine, 0.1% Triton X-100. For elution this buffer with an additional 10 mM ATP was used. Expression and purification of the HSV 1 TK was monitored by SDS-PAGE. SDS-polyacrylamide electrophoresis was carried out either in horizontal gels using the PhastSystem from Pharmacia (with PhastGel Gradient lo-15 and PhastGel SDS buffer strips), or in vertical gels as described by Laemmli (1970).
3. Results and discussion A bacterial vector was constructed that enables both, the preparation of a single-stranded DNA template of the gene of interest and the expression of the corresponding protein as fusion protein. The constructed plasmid pGEX2T-fl contained the intergenic region of the filamentous bacteriophage fl (Fig. 1). According to the orientation of the inserted fl fragment superinfection of bacteria harboring this plasmid with the helper virus M13K07 resulted in the production of single-stranded sense or antisense copies of the plasmid DNA. Additionally the constructed
fl * ori
fl ori
480Q2I IDI/
pBR322 ori Fig. I. Construction of the vectors pGEX2T-fl* and pCEX2T-TKfl +. The origin of replication of the filamentous phage fl (fl ori) was blunt-end cloned into the Asp1 site of the pGEX2T vector yielding the pCEX2T-fl’ vector. In compliance with the open reading frame the gene encoding the HSV 1 TK was cloned into the multiple cloning site of pGEX2T-fl + yielding pGEX2T-TKfl ’. Additionally the vector contains the coding regions for the glutathione binding domain of the glutathione S-transferase (GST) and the thrombin recognition site (amino acid sequence LVPRGS) under control of the chimeric tat promotor (p,,,).
vector pGEX2T-fl contained the coding region for the glutathione binding domain of the glutathione S-transferase (GST) under control of the chimeric tat promotor. The coding sequence for a thrombin recognition site and a polycloning site were adjacent. Thus, a protein of interest cloned in frame into this polycloning site is expressed as N-terminal fusion with GST. Glutathione affinity chromatography and subsequent cleavage with thrombin provides the unfused recombinant protein. First we tested this new phagemid on its capabilities to produce template DNA and to overexpress GST. Preparation of single-stranded DNA and subsequent dideoxy sequencing according to standard methods (Sambrook et al., 1989) was carried out successfully. An oligonucleotide that annealed to the single-stranded template DNA derived from pGEX2T-fl+ just upstream the polycloning site was used as a sequencing primer for all recombinant pGEX vectors. As shown by SDS-PAGE the addition of IPTG to a culture of E. coli 7 I / 18 harboring pGEX2T-fl + resulted in the expression of large quantities of a protein with a molecular weight of 28 kDa which is in agreement with GST (Fig. 2). Thus, the new vector behaves as desired. Then we used this new phagemid for site-directed mutagenesis, sequencing and expression of Herpes Simplex Virus type 1 thymidine kinase (HSV 1 TK). For this
M. Michael et al./Phatmaceutica
142 1
2
3
kDa
M
-
66
-
4.5
-
31
-
21
-
14
Fig. 2. Expression of the GST part of the fusion protein. Gene expression was induced by the addition of IPTG to a bacterial culture of E. coli harboring the phagemid pCEX2T-fl + and monitored by SDS-PAGE. Whole protein of bacteria from 0.66 ~1 of non-induced (lane 1) and induced (lane 2) culture as well as 0.66 ~1 of the supematant (lane 3) containing the soluble protein constituent part of the cells were taken. Samples were solubilised in 2.5% SDS and boiled in the presence of 2-mercaptoethanol for 5 min before electrophoresis on a SDS-polyacrylamide gel using the PhastSystem from Pharmacia. Proteins were visualised by Coomassie Blue staining. The sizes &Da) of the molecular weight markers (lane M) are indicated.
purpose the wildtype tk gene was cloned in frame into the polycloning site of pGEX2T-fl + yielding pGEX2T-TKfl + as shown in Fig. 1. Preparation of single-stranded DNA and subsequent dideoxy sequencing of the wildtype tk gene using the above mentioned pGEX specific primer was carried out successfully. Additionally, both mutagenic oligonucleotides synthesized for site-directed mutagenesis were used as sequencing primers in order to check their correct annealing. Both annealed correctly and dideoxy sequencing was carried out without any problems. As the first step in oligonucleotide-directed mutagenesis is an annealing, priming and extension reaction analogous to dideoxy sequencing, we expected site-directed mutagenesis with our newly constructed phagemid being successful, despite the fact that most single-stranded based mutagenesis protocols recommended M 13 vectors. However, first attempts with our phagemid system resulted in extremely low mutagenic efficiency. The mutagenesis reaction was therefore analysed step by step. A sample was taken at each stage of the mutagenesis procedure and separated by electrophoresis on an agarose gel. The bands on the agarose gel can be assigned to the different forms of DNA produced during the mutagenesis procedure. After the first step most of the template DNA has been converted to a mixture of relaxed RF DNA and nicked DNA indicating that the mutant heteroduplex was generated
Acta Heluetiae 72 (1997) 139-143
successfully. However, the second step in the mutagenesis reaction, which is the complete removal of remaining single-stranded wildtype material, was not sufficient, resulting in a considerably high non-mutant background. This could be due to a minor quality of our single-stranded template DNA, which means that there is too much of co-purified single-stranded DNA of the helper phage. Hence the capacity limit of the T5 exonuclease digestion was reached. These findings confirm that our newly constructed phagemid system is suitable for oligonucleotidedirected mutagenesis but suggest some minor adjustments of the mutagenesis reaction protocol in order to improve the mutagenic efficiency. Expression and purification of the wildtype and the mutated proteins were monitored by SDS-PAGE as shown in Fig. 3. Cytoplasmatic expression of foreign proteins in E. coli often results in the formation of insoluble aggregates. Smith and Johnson (1988) described that most of different GST fusion proteins tested by them were completely or at least partly soluble. However, overexpression of GST-TK fusion protein at 37°C results in inclusion bodies. Attempts to dissolve this aggregates in denaturating agents and refold the protein result in completely inactive preparations. But reduction of the growth tempera-
Ml234567
Fig. 3. SDS-PAGE analysis of expression and purification of HSV 1 TK wildtype and mutants. E. coli harboring the phagemid pGEX2T-TKfl + were grown and harvested as described in Section 2. Whole protein of bacteria from 200 ~1 of induced culture was taken (lane 1). 150 pg of protein from the soluble supematant (lane 2) and 20 pg of glutathione affinity purified fusion protein (lane 3). 20 pg of the purified fusion protein were incubated with 0.02 units thrombin for 3 h (lane 41, for 6 h (lane 5) and for 24 h (lane 6). The completely digested TK was further purified by ATP affinity chromatography (lane 7, 5 kg). Samples were solubilised in 2% SDS and boiled in the presence of 2-mercaptoethanol for 5 min before electrophoresis on a vertical 10% SDS-polyacrylamide gel. Proteins were visualised by Coomassie Blue staining. The sizes &Da) of the molecular weight markers (lane M) are indicated.
M. Michael et al./ Phamaceutica
ture from 37 to 25°C solves this problem, yielding partly soluble GST-TK fusion protein (Fetzer and Folkers, 1992). The fusion proteins can be cleaved by digestion with site-specific proteases, such as thrombin or factor Xa. But often this cleavage is inefficient or non-specific (Dixon et al., 1993). Factor Xa is most frequently the restriction protease of choice, because it cleaves at the C-terminal end of its recognition site resulting in cleavage products with the original first amino acid at the N-terminal end. However, we decided on thrombin because its unmatched stability when dissolved, despite its disadvantage that the cleavage products start always with two additional amino acids Gly-Ser at the N-terminus. We prefer digesting the fusion protein while being bound to the glutathione affinity column. Thus, TK appeared in the eluent while the GST part remained bound to the column. However, thrombin cleavage of the GST-TK fusion protein was surprisingly not site-specific. Thrombin cleaved at two additional and hitherto unknown sites resulting in a truncated TK missing the first 33 amino acids (Fetzer et al., 1994). For final purification ATP affinity chromatography has to be carried out in order to separate the truncated HSV 1 TK from thrombin and the small peptides derived from the non-
Acta Heluetiae 72 (1997) 139-143
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specific stepwise thrombin cleavage. This two-step purification procedure resulted in homogeneously pure and enzymatically active wildtype HSV 1 TK (Fetzer et al., 1994). Both mutants of HSV 1 TK, namely D215R and C25 1G, could be purified by this procedure as well. Quantities of l-2 mg recombinant enzyme per liter of bacterial culture were yielded.
References Dixon, J.E., Hakes, D. and Guan, K.L. (1993) In: Methods in Molecular Genetics, Vol. 2. Academic Press. Fetzer, J. and Folkers, G. (1992) Pharm. Pharmacol. Lett. 2, 112-l 14. Fetzer, J., Michael, M., Bohner, T., Hofbauer, R. and Folkers, G. (1994) Prot. Exp. Purif. 5, 432-441. Laemmli, U.K. (1970) Nature 227, 680-685. Liu, Q. and Summers, W.C. (1988) Virology 163, 638-642. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: a Laboratory Manual, 2nd Ed. CSH Press. Sanger, F., Nicklen, S. and Coulson, A.R. (1977) PNAS (USA) 74, 5463-5467. Smith, D.B. and Johnson, K.S. (1988) Gene 67, 31-40. Taylor, J.W., Ott, J. and Eckstein, F. (1985) Nucleic Acids Res. 13(24), 8765-8785.