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Development of a thermo-regulated expression vector in Escherichia coli B strain
Journal of the Taiwan Institute of Chemical Engineers 53 (2015) 1–5
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Journal of the Taiwan Institute of Chemical Engineers journal homepage: www.elsevier.com/locate/jtice
Development of a thermo-regulated expression vector in Escherichia coli B strain Cheng-Huan Liu a, Po Ting Chen b, Chung-Jen Chiang c, Jei Fu Shaw d, Yun-Peng Chao e,f,∗ a
Department of Food Science and Biotechnology, National Chung Hsing University, Taichung, Taiwan Department of Biotechnology, Southern Taiwan University of Science and Technology, Tainan 710, Taiwan c Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung 40402, Taiwan d Department of Biological Science & Technology, I-Shou University, Kaohsiung City 84001, Taiwan e Department of Chemical Engineering, Feng Chia University, 100 Wenhwa Road, Taichung 40724, Taiwan f Department of Health and Nutrition Biotechnology, Asia University, Taichung 41354, Taiwan b
a r t i c l e
i n f o
Article history: Received 18 November 2014 Revised 26 February 2015 Accepted 26 February 2015 Available online 14 March 2015 Keywords: Thermo-regulated gene expression Recombination protein lacI mutation Expression system Escherichia coli B strain Response surface methodology
a b s t r a c t The lacI gene in the plasmid bearing the T7A1 promoter (PA1 )-driven lacZ was randomly mutated. The mutant library was then screened in Escherichia coli B strain deficient in lacI and lacZ. Based on the LacZ phenotype, one heat-sensitive lacI (lacIts) was isolated and it revealed a mutation with an amino acid substitution, Met42Lys (designated lacI42ts). To examine its performance, the lacI42ts/PA1 -based plasmid was employed for expression of gehC (encoding lipase) in E. coli B strain. Consequently, the strain that received a thermal induction produced 49-fold more GehC in terms of activity than the uninduced level. The expression condition was further optimized, finally leading to a 47% increase in the GehC activity for the strain. Overall, it indicates that the thermo-regulated vector is useful for the recombinant protein production in E. coli B strain. © 2015 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
1. Introduction Enzymes have long been utilized for a variety of industrial applications [1–3]. To obtain an enzyme of interest, the most common practice is to clone the encoded gene on a multi-copy-number plasmid. Transformed with the gene-borne plasmid, the host cell is cultured and then induced for the protein production. This typical approach usually leads to encouraging results and becomes an indispensable method for mass production of recombinant enzymes [4]. Escherichia coli has been regarded as the workhorse of biotechnology and widely applied for the production of recombinant enzymes. A variety of plasmids is available in E. coli [5,6]. Among them, the lac type-derived vectors are most commonly applied for the production of recombinant proteins. These plasmids essentially carry lacI and the lacO site-associated promoter. In the absence of the inducer, the repressor protein (LacI) binds to lacO and the expression of a target gene under control of the promoter is repressed. The target gene is expressed after LacI is inactivated by isopropyl-β -d-thiogalactopyranoside (IPTG), a commonly used inducer. These plasmids are
∗
Corresponding author. Tel.: +886 4 24517250 x3677. E-mail addresses: [email protected] (J.F. Shaw), [email protected] (Y.-P. Chao).
easy to operate and, in particular, the on-and-off regulation of the gene expression facilitates the segregation of the bacterial growth phase from the protein production phase. This method has been proven useful to circumvent the problem of metabolic burden resulting from the protein overproduction. However, IPTG is limited for use in certain industrial applications because of its high cost and toxicity [7]. We have tackled the above-mentioned problem by development of thermo-regulated vectors [8]. These plasmids consist of the heatsensitive lacI (lacIts) and the T7 A1 promoter (PA1 ) associated with lacO. Their usefulness has been demonstrated in E. coli K12-derived strains. As illustrated, the cloned genes on these plasmids are stringently regulated under the permissive condition and timely inducible by heat. It is well recognized that E. coli B strain (e.g., BL21) is superior to the K12 strain for the production of recombinant proteins [9]. E. coli B strain lacks two proteases encoded by lon and ompT, which prevents heterologous proteins from breakdown. The strain also produces less acetate on glucose compared to the E. coli K12 strain. Acetate is known to be very toxic and strongly inhibits E. coli growth, consequently reducing the protein production. In this study, a new variant lacIts (lacI42ts) was isolated and characterized. The result illustrates that he lacI42ts-based plasmid is useful for production of recombinant lipase in a thermo-regulated way in BL21 strain.
http://dx.doi.org/10.1016/j.jtice.2015.02.036 1876-1070/© 2015 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
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C.-H. Liu et al. / Journal of the Taiwan Institute of Chemical Engineers 53 (2015) 1–5 Table 1 Strains, plasmids, and primers used in this study. Main characteristics
Sources
E. coli strain BL21 BL21-1 BL21-3 JW0366-1 VJS676
F− dcm gal ompT hsdS(rB − mB − ) as BL21 lacI785::kan as BL21-1 lacZ::cat lacI785::kan as W1485 (argF-lac)U169
Lab collection This study This study [10] Lab collection
Plasmid pBRINT.Cm pA199A-2Z pA199A2-M42KZ pA199A2-187Z pA199A2-265Z pET28a(+) pET-GehCH pA199A2-MGehCH
lacZ::cat lacI PA1 -lacZ bla+ lacI42ts PA1 -lacZ bla+ lacI187ts PA1 -lacZ bla+ lacI265ts PA1 -lacZ bla+ PT7 -trxA bla+ as pET28a(+) PT7 -gehC as pA199A2-M42KZ PA1 -gehC
[12] [8] This study [8] [8] Novagen Co This study This study
Primer CH14001 CH14002 CHO1 CHO35 MgehC-1 MgehC-2 CH13004 CH13015
5 -GTCGTTTTACAACGTCGTGAC-3 5 -TATGGAAACCGTCGATATTCAG-3 5 -TATTCTCGAGGCCGGAAGGCGAAG-3 5 -TAACTCGCGATAATTGCGTTGCGCTCAC-3 5 -GGGGCCATGGAACAAAAACAATATAAAAAT-3 5 -GTGGTGCTCGAGTTTATTTGTTGATGTTAATTGTTC-3 5 -AAGCTTTCAGTGGTGGTGGTGGTGG-3 5 -TCTAGATTTAATTTTGTTTAACTTTAAGAAG-3
Abbreviations: PT7 , the T7 promoter; bla, ampicillin-resistant determinant; kan, kanamycin-resistant determinant; cat, chloramphenicol-resistant determinant.
2. Materials and methods
2.3. Plasmid construction
2.1. Strain construction
The gehC gene encoding lipase was cloned from Staphylococcus epidermidis ATCC 12228 by PCR with primer MgehC-1 and MgehC-2. By NotI-XhoI cleavage, the PCR DNA was incorporated into plasmid pET28a(+) to obtain plasmid pET-GehCH in strain DH5α . Moreover, gehC was amplified from plasmid pET-GehCH by PCR with primer CH13004 and CH13015. The lacZ in plasmid pA199A2-M42K was removed by the HindIII-XbaI digestion and replaced with the PCR DNA containing gehC. Consequently, the DNA construction resulted in plasmid pA199A2-MGehCH.
The strains, plasmids, and primers applied in this study were summarized in Table 1. E. coli BL21 strain deficient in lacI was constructed by P1 transduction. In brief, strain JW0366-1 (lacI785::kan) [10] was first infected with phage P1cm. The lysogenic strains were scored for exhibiting the resistance to chloramphenicol (20 μg/ml) at 30 °C. Moreover, the transducing phage particles were obtained by growing lysogenic strains in Luria-Bertani (LB) medium [11] at 30 °C. The cell lysis was induced by raising the temperature to 37 °C. Finally, strain BL21 was infected with the transducing phage particles and screened for resistance to kanamycin (50 μg/ml) at 37 °C. One resulting strain was designated BL21-1. Moreover, the DNA containing lacZ::cat was amplified from plasmid pBRINT.Cm [12] by polymerase chain reaction (PCR) with primer CH14001 and CH14002. The PCR DNA was then integrated into strain BL21-1 by electroporation following our reported method [13]. The resulting strain exhibiting resistance to chloramphenicol was renamed as BL21-3. 2.2. Random mutation of lacI The mutant lacI was obtained in several steps. First, the DNA containing lacI was amplified by PCR from plasmid pA199A-2Z [14] with primer CHO1 and CHO35. The error-prone PCR (erPCR) was then conducted using the first-round PCR DNA as the template and the same primers. After cleavage with XhoI and NruI, lacI in plasmid pA199A-2Z was removed and replaced by the erPCR DNA. The resulting plasmid was transformed into strain BL21-3 and transformants were plated on LB agars supplemented with 30 μg/ml ampicillin (Ap) and 20 μg/ml 5-bromo-4-chloro-3-indolyl-β -d-galactopyranoside (X-gal) at 30 °C. Next day, cell colonies appearing on agar plates were transferred to LB plus Ap and X-gal agars by replica plating. Replica agars were incubated at 37 °C or higher overnight. Cell colonies were chosen for exhibiting white colors at 30 °C and blue colors at 37 °C. Finally, one plasmid (designated pA199A2-M42KZ) that conferred on the strain the heat-inducible phenotype was scored and further sequenced for the mutation site of lacI.
2.4. Bacterial culturing and enzyme activity assay Recombinant β -galactosidase and lipase were expressed in strain BL21-3 and BL21-1, respectively. The bacterial growth was measured turbidimetrically at 550 nm (OD550 ). Bacterial strains with plasmids were cultured in shake flasks (250 ml) containing 20 ml LB medium with ampicillin. With an initial cell density at OD550 of 0.08, bacterial cultures were maintained in an incubator set at 30 °C and 200 rpm. Induction was conducted by shifting up the temperature when the cell density reaching the indicated OD550 . After thermal induction for 6 h, bacterial cultures were harvested by centrifugation. The LacZ activity in terms of Miller units was determined as reported previously [8]. To determine the lipase activity, cells were suspended in 0.1 M phosphate buffer (pH 7.4) and disrupted by sonication. Followed by centrifugation, the supernatant was recovered and measured by the Protein assay dye (Bio-Rad Co.). The reaction solution (0.5 ml) consisted of 0.5 mM p-nitrophenyl butyrate, and 50 mM phosphate buffer (pH 6.0). Crude proteins (20 μg) were added to start the reaction at 33 °C for 3 min. The reaction solution was measured at the wave length of 405 nm and the lipase activity was then calculated using the extinction coefficient (18380 M−1 ). The unit (U) of lipase activity was defined 1 μmol p-nitrophenol released/min. 2.5. Optimization of the production condition The production condition for recombinant lipase was optimized by using the central composite design (CCD) of the response surface
C.-H. Liu et al. / Journal of the Taiwan Institute of Chemical Engineers 53 (2015) 1–5
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Table 2 Thermo-induced production of LacZ. °C
pA199A2-187Z (Miller unit)
30 33 35 37 39 40 42
1386 1790 2240 4276 17,586 21,916 27,235
± ± ± ± ± ± ±
96 (1) 136 (1.3) 383 (1.6) 713 (2.9) 4700 (12.7) 2614 (15.8) 1495 (19.6)
pA199A2-265Z (Miller unit) 169 157 226 351 235 294 332
± ± ± ± ± ± ±
5 (1) 6 (0.9) 12 (1.3) 15 (2.1) 8 (1.4) 64 (1.7) 40 (1.9)
pA199A2-M42KZ (Miller unit) 856 3468 5668 18,498 27,956 30,622 23,254
± ± ± ± ± ± ±
65 (1) 143 (4.1) 660 (6.6) 1751 (21.6) 1011 (32.6) 2648 (35.8) 2135 (27.2)
Strain BL21-3 bearing each plasmid was cultivated at 30 °C and then shifted to the indicated temperature when the cell density reached OD550 of 0.3. The induced temperature was then maintained throughout the experiment. The number in parenthesis indicates the induction ratio, which is defined as the induced level relative to the expression level at 30 °C. The data with the standard deviation were taken from three independent experiments.
methodology (RSM). According to our previous report [15], the response surface is described by a polynomial model given below:
lacI42ts PA1
W = β0 + β1 Z1 + β2 Z2 + β12 Z1 Z2 + β11 Z12 + β22 Z22 where W, Z, and β stand for the lipase activity, the independent variable, and the regression coefficient, respectively. The main, interaction, and quadratic effect of two independent variables are defined by the respective coefficients. 3. Results and discussion 3.1. Identification of lacI42ts A series of thermo-inducible plasmids has been developed based on plasmid pA199A-2 that contains PA1 coupled with various lacIts [8]. Among them, plasmid pA199A2-265 and pA199A2-187 carry lacI265ts (Gly265Asp substitution) and lacI187ts (Gly187Ser substitution), respectively. The two vectors perform well in thermo-regulated expression of cloned genes in E. coli K-12 strain [8,16]. Moreover, they have been coupled with the runaway-replication plasmid or incorporated into the T7 expression system for effective production of recombinant proteins [17,18]. It was intriguing to investigate their performance in E. coli B strain. Therefore, plasmid pA199A2-187Z (plasmid pA199A2-187 with lacZ clone) and pA199A2-265Z (plasmid pA199A2-265 with lacZ clone) were individually transformed into strain BL21-3 deficient in lacI and lacZ, resulting in strain BL213/pA199A2-187Z and BL21-3/pA199A2-265Z. Shake-flask cultures of the two strains were carried out and induced thermally for the protein production. Unexpectedly, the LacZ level in strain BL21-3/pA199A2265Z was not inducible by heat (Table 2). In contrast, strain BL21-3/pA199A2-187Z produced the maximum LacZ when the temperature was shifted from 30 °C to 42 °C (30→42 °C). This maximum production is 20 folds more than that at 30 °C, which accounts for the induction ratio of 20. However, this induction ratio in B strain is much lower than that in K-12 (ca. 72) [8]. The phenotype of the mutant lacIts is manifested by the ratio of lacI to the lac operator [19]. This phenomenon has been justified by a series of the thermo-regulated plasmid pA199A2-Its, which carries PA1 along with two synthetic lacO sites and a single copy of lacIts [8]. Using LacZ as a reporter, the plasmid bearing either lacI265ts or lacI187ts features with stringency (i.e., low basal level at 30 °C) and high expression level upon thermal induction in E. coli K12 strain [8]. However, their performance is either poor or ineffective in E. coli B strain as shown above. It implies that these mutant lacIts likely assume a distinct conformation in E. coli K12 and B strain, which in turn determines their thermo-sensitive feature. Next, we turned to search for a new lacIts applicable in E. coli B strain. This work was carried out by random mutation of lacI on plasmid pA199A-2Z (plasmid pA199A-2 with lacZ clone). Strain BL21-3 was used for selection of the mutant lacI. Over 5000 cell colonies were
pMB1 ori
pA199A2-M42KZ lacZ bla rrnB T1T2
Fig. 1. Schematic drawing of plasmid pA199A2-M42KZ. Shown were the essential features of plasmid pA199A2-M42KZ. Plasmid pA199A2-MGehCH is similar to plasmid pA199A2-M42KZ with lacZ replaced by gehC. Abbreviations: pMB1 ori, pMB1 origin of replication; rrnB T1T2, rrnBT1/T2 terminator. Refer to text and Table 1 footnote for others.
screened for the desired trait. Among them, one plasmid (pA199A2M42KZ) in the strain was found to exhibit the lacIts phenotype. As analyzed by the DNA sequencing, the mutation site of lacI in plasmid pA199A2-M42KZ was identified with a Met42Lys substitution. Moreover, plasmid pA199A2-M42KZ was recovered and re-introduced into strain BL21-3. The plasmid-carrying strain (BL21-3/pA199A2-M42KZ) was grown and induced thermally for the LacZ production. As a result, the LacZ level in the strain greatly increased once the induction temperature exceeded 37 °C. The maximum LacZ level was obtained with the thermal induction at 40 °C (Table 2), leading to the induction ratio of 36. In addition, the maximum LacZ level (induced at 40 °C) in strain BL21-3/pA199A2-M42KZ is 11% higher than that (induced at 42 °C) in strain BL21-3/pA199A2-187Z. Overall, the lacI42ts/PA1 -based plasmid (Fig. 1) outperforms the lacI187ts/PA1 -based one in that it exhibits a lower basal level and a higher expression level at a lower induction temperature in E. coli B strain.
3.2. Regulated production of lipase by lacI42ts S. epidermidis gehC encodes lipase which enables efficient synthesis of various flavor esters in the aqueous solution [20]. The usefulness of the lacI42ts/PA1 -based plasmid was further investigated for production of recombinant GehC. This was carried out by construction of plasmid pA199A2-MGehCH carrying gehC under control of the lacI42ts-regulated PA1 . Plasmid pA199A2-MGehCH was transformed into strain BL21-1 lacking lacI. As a result, the GehC production in terms of the enzyme activity increased with the increasing induction temperature and reached the maximum by the 30→40 °C induction (Fig. 2). The maximum GehC activity is 49-fold higher than that at 30 °C.
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C.-H. Liu et al. / Journal of the Taiwan Institute of Chemical Engineers 53 (2015) 1–5 Table 3 Optimization of the production condition for GehC by the CCD method. Trail
1 2 3 4 5 6 7 8 9 10 11 12 13 Fig. 2. The thermo-induced production of recombinant GehC. Strain BL21-l or VJS676 with plasmid pA199A2-MGehCH was cultivated in a shake flask at 30 °C. The protein production was induced by shifting the bacterial culture to the indicated temperature at OD550 of 0.3. The induction ratio for the GehC production was defined as the activity at each induced temperature relative to that at 30 °C. For reference, the GehC activity for each strain at various temperatures was shown in Supplementary Fig. 1.
It was intriguing to see how this system performed in E. coli K-12 strain. Therefore, plasmid pA199A2-MGehCH was transformed into lacI-null strain VJS676 [18]. The plasmid-carrying strain (VJS676/pA199A2-MGehCH) was cultured in a similar manner and induced for the GehC production. Consequently, the expression of GehC in the strain was unregulated (Fig. 2). It indicates that LacI with M42K lacks its repressor activity in E. coli K-12 strain. As illustrated with the GehC production above, the lacI42ts/PA1 based plasmid in E. coli B strain exhibited tight regulation and high expression upon thermal induction. In contrast, the plasmid was not thermally inducible in E. coli K12 strain (Fig. 2). As revealed from the crystal structure, LacI is a homotetramer with a single subunit consisting of 360 amino acids [21]. It consists of two domains responsible for the operator binding and inducer recognition, respectively. The former domain comprises the first 59 N-terminal amino acids while the latter involves the rest residues of LacI. Note that lacI42ts has the mutation site in the operator-binding domain while mutation residues of lacI187ts and lacI265ts are located in the inducer-recognition domain. In general, these mutant lacIts seem to display a straindependent phenotype. However, the correlation of the strain-specific phenotype with the mutation site in the LacI domains still remains elusive. 3.3. Optimization of the GehC production The induction cell density (OD) and the induction temperature appear to be the key factors determining the efficiency of the lacI42ts/PA1 -based expressing system [8]. Therefore, the production condition for GehC was optimized by the CCD of the RSM method. As listed in Table 3, 13 experimental trials were carried out with three levels for each variable. The result of ANOVA for the polynomial model was summarized in Table 4 and described by the following equation:
W = 2.114 + 0.097Z1 + 0.222Z2 − 0.07Z1 Z2 − 0.381Z12 − 0.526Z22 The goodness of fit for this model was supported by the determination coefficient (R2 = 0.98). As indicated by the Fisher’s F-test, the effect of and the quadratic effect of induction OD (P = 0.00185 and P < 0.0001) and the induction temperature (P = 0.0002 and P < 0.0001) were statistically significant. This suggests that the two factors play the determining role in the GehC production by the thermo-regulated expression system. Fig. 3 shows the response surface governed by the two independent variables. The model predicts that the strain
Coded levelsa
Actual levels
Z1
Z2
Z1 (OD550 )
Z2 (°C)
0 0 0 1 0 0 0 −1 1 0 1 −1 −1
0 0 1 1 0 0 0 1 0 −1 −1 0 −1
0.6 0.6 0.6 0.9 0.6 0.6 0.6 0.3 0.9 0.6 0.9 0.3 0.3
37 37 42 42 37 37 37 42 37 32 32 37 32
Total activity (U)
2.13 2.12 1.88 1.38 2.11 2.12 2.12 1.41 1.90 1.27 1.16 1.54 0.91
a Three levels for each factor were used. Symbols: −1, low level; 0, middle level; 1, high level.
Table 4 Result of ANOVA for the CCD method. Factor
Coefficient estimate
Standard error
P-value
Intercept Z1 Z2 Z1 × Z2 Z1 × Z1 Z2 × Z2
2.114 0.097 0.222 −0.07 −0.381 −0.526
0.033 0.032 0.032 0.039 0.047 0.047
<0.0001 0.00185 0.0002 0.1285 <0.0001 <0.0001
3 .0
2 .5 2 .0
U
1 .5
1 .0 0 .5 44 42
40 38
Z2
36 34
32
3 0 0 .2
0 .3
0.4
0 .5
0 .6
0 .7
0 .8
0.9
1.0
Z1
Fig. 3. Response surface plot. The plot shows the effect of factor Z1 (induction OD) and Z2 (induction temperature) on the GehC production.
produces the maximum GehC with the total activity reaching 2.15 U when the induction is administrated at 38 °C and OD of 0.6. The real experiment was conducted under the optimization condition and resulted in the enzyme activity of 2.16 U, indicating the applicability of this model. This GehC production is 47% more than the maximum level (ca. 1.47 U) obtained without optimization (Fig. 2).
4. Conclusions In this work, we isolated a new mutant lacI42ts that functioned in E. coli B strain. As illustrated here, the expression system carrying lacI42ts coupled with PA1 is useful for thermo-regulated expression of the cloned gene. The gene expression level can be further optimized by adjusting the induction OD and the induction temperature as determined by the CCD of RSM. In addition, the lacI42ts/PA1 -based module has other potential applications. For instance, the system by incorporation of this module into the runaway-replication plasmid [17,22] or the T7 expression system [18] is expected to work more efficiently for protein overproduction in E. coli B strain.
C.-H. Liu et al. / Journal of the Taiwan Institute of Chemical Engineers 53 (2015) 1–5
Acknowledgments This work is supported by Ministry of Science and Technology, Taiwan (NSC 101-2221-E-035-057-MY3 and MOST 104-2622-E-035011-CC1 & 104-2623-E-035-004-ET). Supplementary materials Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.jtice.2015.02.036. References [1] Ogawa J, Shimizu S. Microbial enzymes: new industrial applications from traditional screening methods. Trends Biotechnol 1999;17:13. [2] Jegannathan KR, Nielsen PH. Environmental assessment of enzyme use in industrial production – a literature review. J Cleaner Prod 2013;42:228. [3] Munir K, Maqsood S. A review on role of exogenous enzyme supplementation in poultry. Emir J Food Agric 2013;25:66. [4] Sorensen HP, Mortensen KK. Advanced genetic strategies for recombinant protein expression in Escherichia coli. J Biotechnol 2005;115:113. [5] Makrides SC. Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol Rev 1996;60:512. [6] Saida F, Uzand M, Odaert B, Bontems F. Expression of highly toxic genes in E. coli: special strategies and genetic tools. Curr Protein Pept Sci 2006;7:47. [7] Figge J, Wright C, Collins CJ, Robberts TM, Livingston DM. Stringent regulation of stably integrated chloramphenicol acetyl transferase genes by E. coli lac repressor in monkey cells. Cell 1988;52:713. [8] Chao YP, Chern JT, Wen CS, Fu H. Construction and characterization of thermoinducible vectors derived from heat-sensitive lacI genes in combination with the T7 A1 promoter. Biotechnol Bioeng 2002;79:1.
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[9] Waegeman H, Soetaert W. Increasing recombinant protein production in Escherichia coli through metabolic and genetic engineering. J Ind Microbiol Biotechnol 2011;38:1891. [10] Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2006;2:0008. [11] Miller JH. Experiments in molecular genetics. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory; 1972. [12] Balbas P, Alexeyev M, Shokolenko I, Bolivar F, Valle F. A pBRINT family of plasmids for integration of cloned DNA into the Escherichia coli chromosome. Gene 1996;172:65. [13] Chiang CJ, Saini M, Lee HM, Wang ZW, Chen PT, Chao YP. Genomic engineering of Escherichia coli by the phage attachment site-based integration system with mutant loxP sites. Process Biochem. 2012;47:2246. [14] Chao YP, Chiang CJ, Wang YL, Wang ZW. Applicability of new expression vectors for both engineering uses and biological studies. Biotechnol Prog 2003;19:1076. [15] Chiang CJ, Chen PT, Yeh CY, Chao YP. Statistical optimization of one-step immobilization process for recombinant endoglucanase from Clostridium thermocellum. Process Biochem. 2013;47:2246. [16] McCabe KM, Lacherndo EJ, Albino-Flores I, Sheehan E, Hernandez M. LacI(Ts)regulated expression as an in situ intracellular biomolecular thermometer. Appl Environ Microbiol 2011;77:2863. [17] Chao YP, Wen CS, Wang JY. A facile and efficient method to achieve LacZ overproduction by the expression vector carrying the thermoregulated promoter and plasmid copy number. Biotechnol Prog 2001;20:420. [18] Wang ZW, Law WS, Chao YP. Improvement of the thermoregulated T7 expression system by using the heat-sensitive lacI. Biotechnol Prog 2004;20:1352. [19] Hasan N, Szybalski W. Construction of lacIts and lacIq ts expression plasmids and evaluation of the thermosensitive lac repressor. Gene 1995;163:35. [20] Chang RC, Chou SJ, Shaw JF. Synthesis of fatty acid esters by recombinant Staphylococcus epidermidis lipases in aqueous environment. J Agric Food Chem 2001;49:2619. [21] Beyreuther K, Adler K, Geisler N, Klemm A. The amino-acid sequence of lac repressor. Proc Natl Acad Sci USA 1973;70:3576. [22] Chao YP, Lai ZJ, Lo TE. Use of lac fusion to approach the high TyrB production with a runaway-replication vector. J Chin Inst Chem Eng 2000;31:157.
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Journal of the Taiwan Institute of Chemical Engineers journal homepage: www.elsevier.com/locate/jtice
Development of a thermo-regulated expression vector in Escherichia coli B strain Cheng-Huan Liu a, Po Ting Chen b, Chung-Jen Chiang c, Jei Fu Shaw d, Yun-Peng Chao e,f,∗ a
Department of Food Science and Biotechnology, National Chung Hsing University, Taichung, Taiwan Department of Biotechnology, Southern Taiwan University of Science and Technology, Tainan 710, Taiwan c Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung 40402, Taiwan d Department of Biological Science & Technology, I-Shou University, Kaohsiung City 84001, Taiwan e Department of Chemical Engineering, Feng Chia University, 100 Wenhwa Road, Taichung 40724, Taiwan f Department of Health and Nutrition Biotechnology, Asia University, Taichung 41354, Taiwan b
a r t i c l e
i n f o
Article history: Received 18 November 2014 Revised 26 February 2015 Accepted 26 February 2015 Available online 14 March 2015 Keywords: Thermo-regulated gene expression Recombination protein lacI mutation Expression system Escherichia coli B strain Response surface methodology
a b s t r a c t The lacI gene in the plasmid bearing the T7A1 promoter (PA1 )-driven lacZ was randomly mutated. The mutant library was then screened in Escherichia coli B strain deficient in lacI and lacZ. Based on the LacZ phenotype, one heat-sensitive lacI (lacIts) was isolated and it revealed a mutation with an amino acid substitution, Met42Lys (designated lacI42ts). To examine its performance, the lacI42ts/PA1 -based plasmid was employed for expression of gehC (encoding lipase) in E. coli B strain. Consequently, the strain that received a thermal induction produced 49-fold more GehC in terms of activity than the uninduced level. The expression condition was further optimized, finally leading to a 47% increase in the GehC activity for the strain. Overall, it indicates that the thermo-regulated vector is useful for the recombinant protein production in E. coli B strain. © 2015 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
1. Introduction Enzymes have long been utilized for a variety of industrial applications [1–3]. To obtain an enzyme of interest, the most common practice is to clone the encoded gene on a multi-copy-number plasmid. Transformed with the gene-borne plasmid, the host cell is cultured and then induced for the protein production. This typical approach usually leads to encouraging results and becomes an indispensable method for mass production of recombinant enzymes [4]. Escherichia coli has been regarded as the workhorse of biotechnology and widely applied for the production of recombinant enzymes. A variety of plasmids is available in E. coli [5,6]. Among them, the lac type-derived vectors are most commonly applied for the production of recombinant proteins. These plasmids essentially carry lacI and the lacO site-associated promoter. In the absence of the inducer, the repressor protein (LacI) binds to lacO and the expression of a target gene under control of the promoter is repressed. The target gene is expressed after LacI is inactivated by isopropyl-β -d-thiogalactopyranoside (IPTG), a commonly used inducer. These plasmids are
∗
Corresponding author. Tel.: +886 4 24517250 x3677. E-mail addresses: [email protected] (J.F. Shaw), [email protected] (Y.-P. Chao).
easy to operate and, in particular, the on-and-off regulation of the gene expression facilitates the segregation of the bacterial growth phase from the protein production phase. This method has been proven useful to circumvent the problem of metabolic burden resulting from the protein overproduction. However, IPTG is limited for use in certain industrial applications because of its high cost and toxicity [7]. We have tackled the above-mentioned problem by development of thermo-regulated vectors [8]. These plasmids consist of the heatsensitive lacI (lacIts) and the T7 A1 promoter (PA1 ) associated with lacO. Their usefulness has been demonstrated in E. coli K12-derived strains. As illustrated, the cloned genes on these plasmids are stringently regulated under the permissive condition and timely inducible by heat. It is well recognized that E. coli B strain (e.g., BL21) is superior to the K12 strain for the production of recombinant proteins [9]. E. coli B strain lacks two proteases encoded by lon and ompT, which prevents heterologous proteins from breakdown. The strain also produces less acetate on glucose compared to the E. coli K12 strain. Acetate is known to be very toxic and strongly inhibits E. coli growth, consequently reducing the protein production. In this study, a new variant lacIts (lacI42ts) was isolated and characterized. The result illustrates that he lacI42ts-based plasmid is useful for production of recombinant lipase in a thermo-regulated way in BL21 strain.
http://dx.doi.org/10.1016/j.jtice.2015.02.036 1876-1070/© 2015 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
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C.-H. Liu et al. / Journal of the Taiwan Institute of Chemical Engineers 53 (2015) 1–5 Table 1 Strains, plasmids, and primers used in this study. Main characteristics
Sources
E. coli strain BL21 BL21-1 BL21-3 JW0366-1 VJS676
F− dcm gal ompT hsdS(rB − mB − ) as BL21 lacI785::kan as BL21-1 lacZ::cat lacI785::kan as W1485 (argF-lac)U169
Lab collection This study This study [10] Lab collection
Plasmid pBRINT.Cm pA199A-2Z pA199A2-M42KZ pA199A2-187Z pA199A2-265Z pET28a(+) pET-GehCH pA199A2-MGehCH
lacZ::cat lacI PA1 -lacZ bla+ lacI42ts PA1 -lacZ bla+ lacI187ts PA1 -lacZ bla+ lacI265ts PA1 -lacZ bla+ PT7 -trxA bla+ as pET28a(+) PT7 -gehC as pA199A2-M42KZ PA1 -gehC
[12] [8] This study [8] [8] Novagen Co This study This study
Primer CH14001 CH14002 CHO1 CHO35 MgehC-1 MgehC-2 CH13004 CH13015
5 -GTCGTTTTACAACGTCGTGAC-3 5 -TATGGAAACCGTCGATATTCAG-3 5 -TATTCTCGAGGCCGGAAGGCGAAG-3 5 -TAACTCGCGATAATTGCGTTGCGCTCAC-3 5 -GGGGCCATGGAACAAAAACAATATAAAAAT-3 5 -GTGGTGCTCGAGTTTATTTGTTGATGTTAATTGTTC-3 5 -AAGCTTTCAGTGGTGGTGGTGGTGG-3 5 -TCTAGATTTAATTTTGTTTAACTTTAAGAAG-3
Abbreviations: PT7 , the T7 promoter; bla, ampicillin-resistant determinant; kan, kanamycin-resistant determinant; cat, chloramphenicol-resistant determinant.
2. Materials and methods
2.3. Plasmid construction
2.1. Strain construction
The gehC gene encoding lipase was cloned from Staphylococcus epidermidis ATCC 12228 by PCR with primer MgehC-1 and MgehC-2. By NotI-XhoI cleavage, the PCR DNA was incorporated into plasmid pET28a(+) to obtain plasmid pET-GehCH in strain DH5α . Moreover, gehC was amplified from plasmid pET-GehCH by PCR with primer CH13004 and CH13015. The lacZ in plasmid pA199A2-M42K was removed by the HindIII-XbaI digestion and replaced with the PCR DNA containing gehC. Consequently, the DNA construction resulted in plasmid pA199A2-MGehCH.
The strains, plasmids, and primers applied in this study were summarized in Table 1. E. coli BL21 strain deficient in lacI was constructed by P1 transduction. In brief, strain JW0366-1 (lacI785::kan) [10] was first infected with phage P1cm. The lysogenic strains were scored for exhibiting the resistance to chloramphenicol (20 μg/ml) at 30 °C. Moreover, the transducing phage particles were obtained by growing lysogenic strains in Luria-Bertani (LB) medium [11] at 30 °C. The cell lysis was induced by raising the temperature to 37 °C. Finally, strain BL21 was infected with the transducing phage particles and screened for resistance to kanamycin (50 μg/ml) at 37 °C. One resulting strain was designated BL21-1. Moreover, the DNA containing lacZ::cat was amplified from plasmid pBRINT.Cm [12] by polymerase chain reaction (PCR) with primer CH14001 and CH14002. The PCR DNA was then integrated into strain BL21-1 by electroporation following our reported method [13]. The resulting strain exhibiting resistance to chloramphenicol was renamed as BL21-3. 2.2. Random mutation of lacI The mutant lacI was obtained in several steps. First, the DNA containing lacI was amplified by PCR from plasmid pA199A-2Z [14] with primer CHO1 and CHO35. The error-prone PCR (erPCR) was then conducted using the first-round PCR DNA as the template and the same primers. After cleavage with XhoI and NruI, lacI in plasmid pA199A-2Z was removed and replaced by the erPCR DNA. The resulting plasmid was transformed into strain BL21-3 and transformants were plated on LB agars supplemented with 30 μg/ml ampicillin (Ap) and 20 μg/ml 5-bromo-4-chloro-3-indolyl-β -d-galactopyranoside (X-gal) at 30 °C. Next day, cell colonies appearing on agar plates were transferred to LB plus Ap and X-gal agars by replica plating. Replica agars were incubated at 37 °C or higher overnight. Cell colonies were chosen for exhibiting white colors at 30 °C and blue colors at 37 °C. Finally, one plasmid (designated pA199A2-M42KZ) that conferred on the strain the heat-inducible phenotype was scored and further sequenced for the mutation site of lacI.
2.4. Bacterial culturing and enzyme activity assay Recombinant β -galactosidase and lipase were expressed in strain BL21-3 and BL21-1, respectively. The bacterial growth was measured turbidimetrically at 550 nm (OD550 ). Bacterial strains with plasmids were cultured in shake flasks (250 ml) containing 20 ml LB medium with ampicillin. With an initial cell density at OD550 of 0.08, bacterial cultures were maintained in an incubator set at 30 °C and 200 rpm. Induction was conducted by shifting up the temperature when the cell density reaching the indicated OD550 . After thermal induction for 6 h, bacterial cultures were harvested by centrifugation. The LacZ activity in terms of Miller units was determined as reported previously [8]. To determine the lipase activity, cells were suspended in 0.1 M phosphate buffer (pH 7.4) and disrupted by sonication. Followed by centrifugation, the supernatant was recovered and measured by the Protein assay dye (Bio-Rad Co.). The reaction solution (0.5 ml) consisted of 0.5 mM p-nitrophenyl butyrate, and 50 mM phosphate buffer (pH 6.0). Crude proteins (20 μg) were added to start the reaction at 33 °C for 3 min. The reaction solution was measured at the wave length of 405 nm and the lipase activity was then calculated using the extinction coefficient (18380 M−1 ). The unit (U) of lipase activity was defined 1 μmol p-nitrophenol released/min. 2.5. Optimization of the production condition The production condition for recombinant lipase was optimized by using the central composite design (CCD) of the response surface
C.-H. Liu et al. / Journal of the Taiwan Institute of Chemical Engineers 53 (2015) 1–5
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Table 2 Thermo-induced production of LacZ. °C
pA199A2-187Z (Miller unit)
30 33 35 37 39 40 42
1386 1790 2240 4276 17,586 21,916 27,235
± ± ± ± ± ± ±
96 (1) 136 (1.3) 383 (1.6) 713 (2.9) 4700 (12.7) 2614 (15.8) 1495 (19.6)
pA199A2-265Z (Miller unit) 169 157 226 351 235 294 332
± ± ± ± ± ± ±
5 (1) 6 (0.9) 12 (1.3) 15 (2.1) 8 (1.4) 64 (1.7) 40 (1.9)
pA199A2-M42KZ (Miller unit) 856 3468 5668 18,498 27,956 30,622 23,254
± ± ± ± ± ± ±
65 (1) 143 (4.1) 660 (6.6) 1751 (21.6) 1011 (32.6) 2648 (35.8) 2135 (27.2)
Strain BL21-3 bearing each plasmid was cultivated at 30 °C and then shifted to the indicated temperature when the cell density reached OD550 of 0.3. The induced temperature was then maintained throughout the experiment. The number in parenthesis indicates the induction ratio, which is defined as the induced level relative to the expression level at 30 °C. The data with the standard deviation were taken from three independent experiments.
methodology (RSM). According to our previous report [15], the response surface is described by a polynomial model given below:
lacI42ts PA1
W = β0 + β1 Z1 + β2 Z2 + β12 Z1 Z2 + β11 Z12 + β22 Z22 where W, Z, and β stand for the lipase activity, the independent variable, and the regression coefficient, respectively. The main, interaction, and quadratic effect of two independent variables are defined by the respective coefficients. 3. Results and discussion 3.1. Identification of lacI42ts A series of thermo-inducible plasmids has been developed based on plasmid pA199A-2 that contains PA1 coupled with various lacIts [8]. Among them, plasmid pA199A2-265 and pA199A2-187 carry lacI265ts (Gly265Asp substitution) and lacI187ts (Gly187Ser substitution), respectively. The two vectors perform well in thermo-regulated expression of cloned genes in E. coli K-12 strain [8,16]. Moreover, they have been coupled with the runaway-replication plasmid or incorporated into the T7 expression system for effective production of recombinant proteins [17,18]. It was intriguing to investigate their performance in E. coli B strain. Therefore, plasmid pA199A2-187Z (plasmid pA199A2-187 with lacZ clone) and pA199A2-265Z (plasmid pA199A2-265 with lacZ clone) were individually transformed into strain BL21-3 deficient in lacI and lacZ, resulting in strain BL213/pA199A2-187Z and BL21-3/pA199A2-265Z. Shake-flask cultures of the two strains were carried out and induced thermally for the protein production. Unexpectedly, the LacZ level in strain BL21-3/pA199A2265Z was not inducible by heat (Table 2). In contrast, strain BL21-3/pA199A2-187Z produced the maximum LacZ when the temperature was shifted from 30 °C to 42 °C (30→42 °C). This maximum production is 20 folds more than that at 30 °C, which accounts for the induction ratio of 20. However, this induction ratio in B strain is much lower than that in K-12 (ca. 72) [8]. The phenotype of the mutant lacIts is manifested by the ratio of lacI to the lac operator [19]. This phenomenon has been justified by a series of the thermo-regulated plasmid pA199A2-Its, which carries PA1 along with two synthetic lacO sites and a single copy of lacIts [8]. Using LacZ as a reporter, the plasmid bearing either lacI265ts or lacI187ts features with stringency (i.e., low basal level at 30 °C) and high expression level upon thermal induction in E. coli K12 strain [8]. However, their performance is either poor or ineffective in E. coli B strain as shown above. It implies that these mutant lacIts likely assume a distinct conformation in E. coli K12 and B strain, which in turn determines their thermo-sensitive feature. Next, we turned to search for a new lacIts applicable in E. coli B strain. This work was carried out by random mutation of lacI on plasmid pA199A-2Z (plasmid pA199A-2 with lacZ clone). Strain BL21-3 was used for selection of the mutant lacI. Over 5000 cell colonies were
pMB1 ori
pA199A2-M42KZ lacZ bla rrnB T1T2
Fig. 1. Schematic drawing of plasmid pA199A2-M42KZ. Shown were the essential features of plasmid pA199A2-M42KZ. Plasmid pA199A2-MGehCH is similar to plasmid pA199A2-M42KZ with lacZ replaced by gehC. Abbreviations: pMB1 ori, pMB1 origin of replication; rrnB T1T2, rrnBT1/T2 terminator. Refer to text and Table 1 footnote for others.
screened for the desired trait. Among them, one plasmid (pA199A2M42KZ) in the strain was found to exhibit the lacIts phenotype. As analyzed by the DNA sequencing, the mutation site of lacI in plasmid pA199A2-M42KZ was identified with a Met42Lys substitution. Moreover, plasmid pA199A2-M42KZ was recovered and re-introduced into strain BL21-3. The plasmid-carrying strain (BL21-3/pA199A2-M42KZ) was grown and induced thermally for the LacZ production. As a result, the LacZ level in the strain greatly increased once the induction temperature exceeded 37 °C. The maximum LacZ level was obtained with the thermal induction at 40 °C (Table 2), leading to the induction ratio of 36. In addition, the maximum LacZ level (induced at 40 °C) in strain BL21-3/pA199A2-M42KZ is 11% higher than that (induced at 42 °C) in strain BL21-3/pA199A2-187Z. Overall, the lacI42ts/PA1 -based plasmid (Fig. 1) outperforms the lacI187ts/PA1 -based one in that it exhibits a lower basal level and a higher expression level at a lower induction temperature in E. coli B strain.
3.2. Regulated production of lipase by lacI42ts S. epidermidis gehC encodes lipase which enables efficient synthesis of various flavor esters in the aqueous solution [20]. The usefulness of the lacI42ts/PA1 -based plasmid was further investigated for production of recombinant GehC. This was carried out by construction of plasmid pA199A2-MGehCH carrying gehC under control of the lacI42ts-regulated PA1 . Plasmid pA199A2-MGehCH was transformed into strain BL21-1 lacking lacI. As a result, the GehC production in terms of the enzyme activity increased with the increasing induction temperature and reached the maximum by the 30→40 °C induction (Fig. 2). The maximum GehC activity is 49-fold higher than that at 30 °C.
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C.-H. Liu et al. / Journal of the Taiwan Institute of Chemical Engineers 53 (2015) 1–5 Table 3 Optimization of the production condition for GehC by the CCD method. Trail
1 2 3 4 5 6 7 8 9 10 11 12 13 Fig. 2. The thermo-induced production of recombinant GehC. Strain BL21-l or VJS676 with plasmid pA199A2-MGehCH was cultivated in a shake flask at 30 °C. The protein production was induced by shifting the bacterial culture to the indicated temperature at OD550 of 0.3. The induction ratio for the GehC production was defined as the activity at each induced temperature relative to that at 30 °C. For reference, the GehC activity for each strain at various temperatures was shown in Supplementary Fig. 1.
It was intriguing to see how this system performed in E. coli K-12 strain. Therefore, plasmid pA199A2-MGehCH was transformed into lacI-null strain VJS676 [18]. The plasmid-carrying strain (VJS676/pA199A2-MGehCH) was cultured in a similar manner and induced for the GehC production. Consequently, the expression of GehC in the strain was unregulated (Fig. 2). It indicates that LacI with M42K lacks its repressor activity in E. coli K-12 strain. As illustrated with the GehC production above, the lacI42ts/PA1 based plasmid in E. coli B strain exhibited tight regulation and high expression upon thermal induction. In contrast, the plasmid was not thermally inducible in E. coli K12 strain (Fig. 2). As revealed from the crystal structure, LacI is a homotetramer with a single subunit consisting of 360 amino acids [21]. It consists of two domains responsible for the operator binding and inducer recognition, respectively. The former domain comprises the first 59 N-terminal amino acids while the latter involves the rest residues of LacI. Note that lacI42ts has the mutation site in the operator-binding domain while mutation residues of lacI187ts and lacI265ts are located in the inducer-recognition domain. In general, these mutant lacIts seem to display a straindependent phenotype. However, the correlation of the strain-specific phenotype with the mutation site in the LacI domains still remains elusive. 3.3. Optimization of the GehC production The induction cell density (OD) and the induction temperature appear to be the key factors determining the efficiency of the lacI42ts/PA1 -based expressing system [8]. Therefore, the production condition for GehC was optimized by the CCD of the RSM method. As listed in Table 3, 13 experimental trials were carried out with three levels for each variable. The result of ANOVA for the polynomial model was summarized in Table 4 and described by the following equation:
W = 2.114 + 0.097Z1 + 0.222Z2 − 0.07Z1 Z2 − 0.381Z12 − 0.526Z22 The goodness of fit for this model was supported by the determination coefficient (R2 = 0.98). As indicated by the Fisher’s F-test, the effect of and the quadratic effect of induction OD (P = 0.00185 and P < 0.0001) and the induction temperature (P = 0.0002 and P < 0.0001) were statistically significant. This suggests that the two factors play the determining role in the GehC production by the thermo-regulated expression system. Fig. 3 shows the response surface governed by the two independent variables. The model predicts that the strain
Coded levelsa
Actual levels
Z1
Z2
Z1 (OD550 )
Z2 (°C)
0 0 0 1 0 0 0 −1 1 0 1 −1 −1
0 0 1 1 0 0 0 1 0 −1 −1 0 −1
0.6 0.6 0.6 0.9 0.6 0.6 0.6 0.3 0.9 0.6 0.9 0.3 0.3
37 37 42 42 37 37 37 42 37 32 32 37 32
Total activity (U)
2.13 2.12 1.88 1.38 2.11 2.12 2.12 1.41 1.90 1.27 1.16 1.54 0.91
a Three levels for each factor were used. Symbols: −1, low level; 0, middle level; 1, high level.
Table 4 Result of ANOVA for the CCD method. Factor
Coefficient estimate
Standard error
P-value
Intercept Z1 Z2 Z1 × Z2 Z1 × Z1 Z2 × Z2
2.114 0.097 0.222 −0.07 −0.381 −0.526
0.033 0.032 0.032 0.039 0.047 0.047
<0.0001 0.00185 0.0002 0.1285 <0.0001 <0.0001
3 .0
2 .5 2 .0
U
1 .5
1 .0 0 .5 44 42
40 38
Z2
36 34
32
3 0 0 .2
0 .3
0.4
0 .5
0 .6
0 .7
0 .8
0.9
1.0
Z1
Fig. 3. Response surface plot. The plot shows the effect of factor Z1 (induction OD) and Z2 (induction temperature) on the GehC production.
produces the maximum GehC with the total activity reaching 2.15 U when the induction is administrated at 38 °C and OD of 0.6. The real experiment was conducted under the optimization condition and resulted in the enzyme activity of 2.16 U, indicating the applicability of this model. This GehC production is 47% more than the maximum level (ca. 1.47 U) obtained without optimization (Fig. 2).
4. Conclusions In this work, we isolated a new mutant lacI42ts that functioned in E. coli B strain. As illustrated here, the expression system carrying lacI42ts coupled with PA1 is useful for thermo-regulated expression of the cloned gene. The gene expression level can be further optimized by adjusting the induction OD and the induction temperature as determined by the CCD of RSM. In addition, the lacI42ts/PA1 -based module has other potential applications. For instance, the system by incorporation of this module into the runaway-replication plasmid [17,22] or the T7 expression system [18] is expected to work more efficiently for protein overproduction in E. coli B strain.
C.-H. Liu et al. / Journal of the Taiwan Institute of Chemical Engineers 53 (2015) 1–5
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