Biotechnology Letters 25: 755–760, 2003.

pared with the initial T7 expression system, indicating   and TTE0085 were 5 -CTC CCA TGG CAA CTT
that the DB box can...

Fig. 1. The map of the expression vector pET-DB. The nucleotide sequence indicates the 100 bp of XbaI-NcoI insert ...

ing of the lac repressor at this site effectively reduces
transcription by T7 RNA polymerase, and suppresses
basal ex...

Fig. 3. SDS-PAGE analysis of expression of chicken muscle adeny-
late kinase, human TF-1 cell apoptosis-related pr...

References                                                             Mitta M, Fang L, Inouye M (1997) Deletion anal...
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  1. 1. Biotechnology Letters 25: 755–760, 2003. 755 © 2003 Kluwer Academic Publishers. Printed in the Netherlands. A vector with the downstream box of the initiation codon can highly enhance protein expression in Escherichia coli Xiaolan Zhang, Peng Guo & Guozhong Jing∗ National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, Beijing 100101, P.R. China ∗ Author for correspondence (Fax: +861064872026; E-mail: Received 21 January 2003; Revisions requested 13 February 2003; Revisions received 12 March 2003; Accepted 13 March 2003 Key words: downstream box, expression vector, initiation codon Abstract An expression vector, pET-DB, with a perfectly matching downstream box of the initiation codon has been con- structed on the basis of the pET system. Any gene of interest can then be inserted into the vector. Four genes were used to test the expression efficiency of the vector. The results show that the vector pET-DB can further increase protein expression level at least up to 35–70% as compared with the initial T7 expression system, indicating that the downstream box can enhance protein expression in Escherichia coli. Introduction ficiency of polysome formation probably due to a translation initiation enhancement, however, DB func- The translation initiation of Escherichia coli mRNAs tions only in the presence of SD sequence, they also is facilitated by a cis element upstream of the initia- noted that a perfectly matching DB, i.e. 15 bases tion codon, called the Shine–Dalgarno (SD) sequence. match with anti-DB sequence within 16S rRNA, is This sequence, which is complementary to the 3 important for enhancement of protein synthesis by end of 16S rRNA, enhances the formation of the DB (Etchegaray et al. 1999b). Recently, Sanchez translation initiation complex of the 30S ribosomal et al. (2000) found that 64% of 130 analyzed genes subunit with mRNA. Sprengart et al. (1990, 1996) from Thermus thermophilus showed such a down- proposed that there is another sequence located the stream box. This consensus sequence was found both downstream of the initiation codon in genes 0.3 and in T. thermophilus mRNAs containing an SD sequence 10 of bacteriophage T7, called the downstream box and in mRNAs lacking the SD sequence. These ob- (DB), which is complementary to bases 1469–1483 servations suggest that an mRNA-rRNA interaction within the Escherichia coli 16S rRNA (anti–DB se- at the level of these downstream sequences could, in quence), and serves as an independent translational some cases, compensate for absent SD boxes or rein- signal. Mutations were found in the E. coli gln gene force interactions with existing ones. pET vectors, on which increased the complementarity of downstream the basis of T7 promoter/RNA polymerase, are much sequences to 16S rRNA and resulted in higher trans- more powerful than others for the highest expression lational activity (Faxen et al. 1991). Although it of foreign proteins in E. coli (Studier et al. 1991). has been debated that the downstream box facili- To examine if the DB sequence can further enhance tates formation of the translation initiation complex protein expression level, a universal expression vector, (O’Connor et al. 1999, Moll et al. 2001), the re- pET-DB, with the perfectly matching DB sequence sults from Inouye and colleagues (Mitta et al. 1997, (Etchegaray et al. 1999b) has been constructed on the Etchegaray et al. 1999a,b) showed that the presence basis of T7 promoter. Four genes were used to test of a DB sequence in cold-shock mRNAs played an the expression efficiency of the vector. The results, important role in translation efficiency. Furthermore, given here, show that pET-DB can further increase these researchers revealed that DB enhances the ef- protein expression level at least up to 35–70% as com-
  2. 2. 756 pared with the initial T7 expression system, indicating and TTE0085 were 5 -CTC CCA TGG CAA CTT that the DB box can enhance protein expression in CAA CTA AAA AAT TAC-3 , 5 -GGA ACC ATG E. coli. The improved vector not only makes recom- GGT TCG ACA GAA AAA CTG AAG CAC-3 , 5 - binant protein highly expressed but also simplifies ATA TAC CAT GGG CCA CCA TCA C-3 , 5 -GGT protein purification procedure. ACC ATG GGT CAT GTA ACA ATA CAG GTA AAT-3 , respectively. The SalI reverse primers for the above PCR products were 5 -GGG TCG ACT TAT Materials and methods TGA CCT GAA TCA GCG-3 , 5 -GCT CAG TCG ACT CAC AGC TTG TCG AGG TAG-3 , 5 -GCA Bacterial strains and plasmids GCC GTC GAC ATT AAT AAT CGT CAT CTT CAT CA-3 , 5 -CTG GTC GAC ATT AAG GTA GTA AGT Escherichia coli DH5α and BL21 (DE3) were used TTA TAT ATC-3 , respectively. The PCR products as the cloning and expression host cells, respectively. were inserted into the NcoI and SalI sites of pET-DB Plasmid pET-30a(+) containing T7 lac promoter and after digestion with NcoI and SalI enzymes, result- T7 transcriptional terminator was as an initial vec- ing in the respective plasmids pET-DB-Staph, pET- tor for construction of the vector with DB sequence. DB-AK, pET-DB-TFAR19, pET-DB–TTE0085. For Plasmids pBVS-1 (Jing et al. 1992), pBV-AK (Jing construction of the corresponding control expression et al. 1997), and pHTFAR19 (Feng et al. 2002), which plasmids (without DB sequence), the above template harbor the genes encoding staphylococcal nuclease of DNAs were also used. The NdeI forward primers for 149 residues, chicken muscle adenylate kinase of 194 the PCR products Staph, AK, TFAR19, and TTE0085 residues, and a human TF-1 cell apoptosis-related pro- were 5 -ACA GCA GCC ATA TGG CAA CTT CAA tein TFAR19 of 125 residues, respectively, were used CTA AAA AAT TAC-3 , 5 -ACA GCA GCC ATA as templates for amplifying the corresponding genes. TGT CTA CAG AAA AAC TGA AGC A-3 , 5 -GAG The genomic DNA isolated from Thermoanaerobacter ATA TAC CAT ATG GGC CAC CAT CAC CAC CAT tengcongensis (Bao et al. 2002) was used as a template C-3 , 5 -GAG ATA TAC CAT ATG CAT GTA ACA for amplifying a gene encoding a hypothetical protein ATA CAG GTA AAT-3 , respectively. The SalI reverse TTE 0085 of 117 residues. primers were the same as the above ones. The PCR products were cleaved with NdeI and SalI, and then DNA manipulation inserted into the same sites of pET-28a(+), resulting in the respective plasmids pET-staph, pET-AK, pET- Restriction endonucleases and T4 DNA ligase were TFAR19, and pET-TTE0085. This manipulation made used as recommended by the manufacturer (Promega). a pair of expression plasmids (e.g., pET-DB-staph and Plasmid DNA isolation and transformation of CaCl2 - pET-staph) comparable, that is, the target genes were treated E. coli cells were carried out by the proce- expressed under the same control conditions including dures described by Sambrook et al. (1989). Plasmid promoter, SD and origin of replication etc., except that construction was performed as follows: a chemi- the DB sequence was absent in the control expression cally synthesized oligonucleotide fragment of 100 bp, vectors. which contain an SD sequence and a perfectly match- ing DB sequence (Etchegaray et al. 1999b) followed Expression of target protein genes in E. coli by a His6 -tag and a thrombin cleavage site cod- ing sequences as shown below in Figure 1, was The E. coli cells [BL21(DE3)] harboring a corre- inserted into the XbaI-NcoI sites of pET-30a(+) vec- sponding expression plasmid were cultured, diluted tor to create pET-DB. To test the DB activity for 50-fold with fresh pre-warmed LB medium supple- enhancement of protein expression, four expression mented with 50 µg kanamycin ml−1 and incubated plasimids, which express staphylococcal nuclease at 37 ◦ C until a turbidity of 0.6 at 600 nm was ob- (Staph), chicken muscle adenylate kinase (AK), hu- tained (approx. 108 cells ml−1 ) prior to the induction man TF-1 cell apoptosis-related protein TFAR19 and by 0.4 mM IPTG. In order to compare the expression a hypothetical protein TTE0085 from T. tengcongen- level of proteins in a pair of expression plasmids as sis, were constructed as follows. Plasmids pBVS-1, described above, the whole cell lysates with same cell pBV-AK, pHTFAR19, and T. tengcongensis genomic density were made at each time point, and subjected DNA were used as template DNAs. The NcoI forward to SDS-15% PAGE as described by Jing et al. (1992). primers for the PCR products Staph, AK, TFAR19,
  3. 3. 757 Fig. 1. The map of the expression vector pET-DB. The nucleotide sequence indicates the 100 bp of XbaI-NcoI insert including SD sequence, DB sequence, His6 tag and thrombin cleavage site coding sequences. PT7 and TT7 indicate T7 promoter and T7 terminator, respectively. Proteins were visualized by staining with Coomassie cleavage mixture was then applied onto the second Brilliant Blue G250 and analyzed by using LabWorks metal chelating chromatography column to remove Software (UVP Bio-Doc-It System). To minimize the the fusion partner. The protein samples at each step experimental error, six pairs of independent experi- were analyzed by using SDS-15% PAGE according to ments were carried out. the Blackshear procedure (Blackshear 1984). Proteins were visualized by staining with Coomassie Brilliant Thrombin cleavage and purification of expression Blue G250. proteins As shown in Figure 1, the chemically synthesized Results and discussion oligonucleotide fragment which encodes 22 amino acid residues including the His6 -tag and thrombin Construction of the expression vector with the DB cleavage site can be fused in-frame to the N-terminal sequence (pET-DB) end of any protein of interest with restriction enzyme NcoI site. In this case, target protein is first expressed As described in Materials and methods, the chemi- as a His6 -tagged protein. For purification of the His6 - cally synthesized oligonucleotide fragment of 100 bp, tagged protein (e.g. His6-tagged staphylococcal nu- which contains an SD sequence and a perfectly match- clease), a metal chelating affinity chromatography ing DB sequence followed by a His6 -tag and a throm- was performed according to the routine procedure de- bin cleavage site coding sequences, was inserted into scribed by Feng et al. (2002). The purified protein was the XbaI and NcoI sites of expression vector pET- then incubated with thrombin (Sigma T4648) in the 30a(+) to yield pET-DB (Figure 1). In this vector, ratio of 10 units of thrombin mg−1 protein in cleav- target gene is expressed under the control of strong age buffer (20 mM Tris/HCl, pH 8, 2.5 mM CaCl2 , bacteriophage T7 promoter; a lac operator sequence 10 mM MgCl2 ) at 4 ◦ C for 8 h. After digestion, the is immediately downstream from the promoter. Bind-
  4. 4. 758 ing of the lac repressor at this site effectively reduces transcription by T7 RNA polymerase, and suppresses basal expression in BL21 (DE3) cells (Dubendorff et al. 1991). This vector also carries its own copy of lacI to ensure that enough repressor is made to titrate all available operator sites. Besides these common control elements found in pET system, there is another important translation signal, the downstream box (DB) downstream of the initiation codon, in the new devel- oped expression vector pET-DB. It is known that DB functions as a translational enhancer and that greater complementarity to the anti-DB improves translational efficiency and that specific base pairings like the first three nucleotides (ATG) of the DB sequence may play an important role for the DB activity (Etchegaray et al. 1999b). In this construction, a perfectly matching DB sequence of 15 bases is placed after SD. There are about 7 bases between SD and DB, and the first three nucleotides ATG of DB are also as the initiation codon for expression of target genes. For simplifying purification of target proteins, a His6 -tag and a spe- cific protease thrombin cleavage coding sequences are fused in frame to DB sequence (Figure 1). Any gene of interest can be fused in frame to the above fusion part- ner with NcoI site. The construction not only makes the vector highly enhance protein expression but also simplifies purification procedure of target proteins. pET-DB vector highly enhances the expression level of target proteins To examine whether pET-DB can enhance the expres- sion level of target proteins, four proteins, i.e. staphy- lococcal nuclease, chicken muscle adenylate kinase, human TF-1 cell apoptosis-related protein TFAR19, and TTE0085 protein of T. tengcongensis, were ex- pressed by using pET-DB and pET-28a(+), respec- tively, as described in Materials and methods. Six pairs of independent experiments were carried out under the same conditions. Figure 2 shows the SDS-PAGE pat- terns of the whole cell lysates from E. coli cells harbor- ing pET-DB-staph and pET-staph, respectively, before Fig. 2. SDS-PAGE analysis of expression of staphylococcal nucle- and after induction by IPTG at different time. There is ase by using different vectors. (A) pET-staph, (B) pET-DB-staph. a great difference in the expression level of staphylo- The PAGE gels were stained with Coomassie Brilliant Blue R250. coccal nuclease between pET-DB-staph and pET-staph From lane 1 to lane 5: whole cell lysate without IPTG induction, IPTG induction for 1 h, 2 h, 3 h, and 4 h, respectively. There during the induction. The statistical data from the in- is no any target protein band to be seen in lane 1, indicating that dependent experiments reveal that the expression level the expression system highly suppresses basal expression of foreign of staphylococcal nuclease is increased from 50% to gene in BL21(DE3) cells. 70% by using pET-DB-staph, as compared with that by using pET-staph. Figure 3 shows the SDS-PAGE patterns of the whole cell lysates from E. coli cells
  5. 5. 759 Fig. 3. SDS-PAGE analysis of expression of chicken muscle adeny- late kinase, human TF-1 cell apoptosis-related protein TFAR19, and TTE0085 protein of T. tengcongensis by using different vectors. Lane 1: pET-DB-AK; lane 2: pET-AK; lane 3: pET-DB-TFAR19; lane 4: pET-TFAR19; lane 5: pET-DB-TTE0085; lane 6: pET-TTE0085. Whole cell lysates were prepared after IPTG induc- tion for 4 h. harboring pET-DB-AK, pET-DB-TFAR19, pET-DB- TTE0085, and their corresponding control expression plasmids after induction by IPTG for 4 h, respectively. Compared with their corresponding control expression vectors, the expression level of chicken muscle adeny- Fig. 4. Purification and thrombin cleavage of His6 -tagged staphylo- late kinase, human TF-1 cell apoptosis-related protein coccal nuclease. Lane 1: standard staphylococcal nuclease sample. Lane 2: Sample after thrombin cleavage at 4 ◦ C for 8 h, showing the TFAR19, and TTE0085 protein is increased 11-fold, fusion partner is completely removed under the conditions described 35%, and 38% by using pET-DB vector, respectively. in the text. Lane 3: His6-tagged staphylococcal nuclease sample Although the expression level increase varies with from the main peak of the metal-chelating affinity chromatography. different gene, all the observations described above demonstrate that the pET-DB as a powerful expres- sion vector can further increase recombinant protein chelating column as described above. It is worth not- expression in E. coli. The results also indicate that DB ing that commercially available thrombin (e.g. Sigma does play an important role in enhancement of foreign T4648) is usually contaminated with some proteins. protein synthesis even in pET system, which always The protease and contaminated proteins can be re- shows the highest expression level of foreign proteins. moved by using FPLC MonoS/Mono Q or FPLC Resources S/Resources Q column (Amersham Bio- Simplifying purification of target protein sciences), which is dependent on the pI value of target proteins. Since target protein is first expressed in the form of His6 -tagged fusion protein by using pET-DB, the fu- sion protein can be first purified by using a routine Acknowledgement metal-chelating affinity chromatography procedure as described in Materials and methods in which staphy- This work was supported by a grant (No. lococcal nuclease as a sample protein (Figure 4). G1999075608) from the China Committee for Science Thrombin cleavage of the fusion protein can be ac- and Technology. complished at 4 ◦ C under the nearly physiological conditions, which will facilitate the recovery of ac- tive proteins. To remove fusion partner, the cleavage reaction mixture can be applied onto the second metal-
  6. 6. 760 References Mitta M, Fang L, Inouye M (1997) Deletion analysis of cspA of Es- cherichia coli: requirement of the AT-rich UP element for cspA transcription and the downstream box in the coding region for its Bao Q, Tian Y, Li W, Xu Z, Xuan Z, Hu S, Dong W, Yang J, Chen cold shock induction. Mol. Microbiol. 26: 321–335. Y, Xue Y, Xu Y, Lai X, Huang L, Dong X, Ma Y, Ling L, Tan H, Moll I, Huber M, Grill S, Sairabi, P Mueller F, Brimacombe R, Lon- Chen R, Wang J, Yu J, Yang H (2002) A complete sequence of dei P, Blasi U (2001) Evidence against an interaction between the the T. tengcongensis genome. Genome Res. 12: 689–700. mRNA downstream box and 16S rRNA in translation initiation. Blackshear PJ (1984) Systems for polyacrylamide gel electrophore- J. Bacteriol. 183: 3499–3505. sis. Meth. Enzymol. 104: 237–255. O’Connor M, Asai T, Squires CL, Dahlberg AE (1999) Enhance- Dubendorff JW, Studier FW (1991) Controlling basal expression in ment of translation by the downstream box does not involve an inducible T7 promoter with lac repressor. J. Mol. Biol. 219: mRNA-rRNA base pairing of mRNA with the penultimate stem 45–59. sequence of 16S rRNA. Proc. Natl. Acad. Sci. USA 96: 8973– Etchegaray JP, Inouye M (1999a) A sequence downstream of the 8978. initiation codon is essential for cold shock induction of cspB in Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Escherichia coli. J. Bacteriol. 181: 5852–5854. Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Etchegargy JP, Inouye M (1999b) Translational enhancement by an Spring Harbor Laboratory Press. element downstream of the initiation codon in Escherichia coli. Sanchez R, Roovers M, Glansdorff N (2000) Organization and J. Biol. Chem. 274: 10079–10085. expression of a Thermus thermophilus arginine cluster: pres- Faxen M, Plumbridge J, Isakson LA (1991) Codon choice and ence of unidentified open reading frames and absence of a potential complementarity between mRNA downstream of the Shine–Dalgarno sequence. J. Bacteriol. 182: 5911–5915. initiation codon and bases 1471–1480 in 16S ribosomal RNA Sprengart ML, Fatscher HP, Fuchs E (1990) The initiation of trans- affects expression of glnS. Nucl. Acids Res. 19: 5247–5251. lation in E. coli: apparent base pairing between the 16S rRNA Feng YM, Zhang YM, Jing GZ (2002) Soluble expression in Es- and downstream sequences of the mRNA. Nucl. Acids Res. 18: cherichia coli, purification and characterization of a human TF-1 1719–1723. cell apoptosis-related protein TFAR19. Protein Exp. Purif. 25: Sprengart ML, Fuchs E, Porter AG (1996) The downstream box: 323–329. an efficient and independent translation initiation signal in Es- Jing EX, Zhou B, Luo J, Zhang HJ (1997) Soluble expression cherichia coli. EMBO J. 15: 665–674. of chicken muscle adenylate kinase in Escherichia coli. Prog. Studier FW, Rosenberg AT, Dunn JJ, Dubendorff JW (1991) Use of Biochem. Biophys. 24: 525–528. T7 RNA polymerase to direct expression of cloned genes. Meth. Jing GZ, Liu LJ, Jiang MY, Zou Q, He RQ (1992) High-level ex- Enzymol. 185: 60–89. pression of staphylococcal nuclease R gene in Escherichia coli. J Biotechnol. 22: 271–282.