Division of Chemistry and Chemical Engineering 210-41, California Institute of Technology, Pasadena, CA 91125, USA
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: directed evolution/galactose oxidase/random mutagenesis/StEP recombination
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
These applications of GOase would benefit from access to enzyme variants that are more stable and active towards non-natural substrates. A prerequisite to enzyme modification by powerful directed evolution methods (Arnold, 1998; Petrounia and Arnold, 2000
) is functional expression in a host organism that permits creation and rapid screening of mutant libraries. Escherichia coli is an excellent host for directed evolution, but does not support functional expression of many important eukaryotic enzymes. To date, all biochemical studies of GOase have been performed on the enzyme obtained from its natural source or from fungal (McPherson et al., 1993
; Xu et al., 2000
) and yeast (Whittaker and Whittaker, 2000
) expression systems not suitable for directed evolution. Expression of GOase in E.coli has been attempted (McPherson et al., 1993
), but functional enzyme was obtained only as a lacZ fusion (Lis and Kuramitsu, 1997
). Biochemical characterization of the E.coli-expressed GOase was not reported. Herein we report on functional expression of GOase in E.coli achieved by directed evolution.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
All chemicals were reagent grade or better. 2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), D-galactose and horseradish peroxidase (HRP) were from Sigma (St Louis, MO). Native Fusarium GOase was obtained from Worthington Biochemical Corporation (Lakewood, NJ). Escherichia coli strain BL21(DE3) and vector plasmid pUC18 were purchased from Novagen (Madison, WI). Restriction enzymes and ligase were obtained from Boehringer Mannheim (Indianapolis, IN), Life Technologies (Grand Island, NY) or New England Biolabs (Beverly, MA).
Bacterial strain and plasmids
Bacterial strain BL21(DE3) was used for cloning and library construction. Plasmid pR3 containing the gene for mature GOase fused to the 5'-end of the lacZ fragment was kindly provided by Dr Howard K. Kuramitsu (Department of Oral Biology, State University of New York at Buffalo). The GOase gene was amplified from pR3 by PCR to introduce a HindIII restriction site followed by an ATG initiation codon immediately upstream from the mature GOase sequence and XbaI site immediately downstream from the stop codon. The PCR product was subcloned into a modified vector pUC18 (containing a double lac promoter and lacking the PstI site) to yield pGAO-036.
Construction of GOase mutant libraries
GOase was expressed in E.coli using plasmid pGAO-036. Two approaches were followed for directed evolution: (A) random mutagenesis of the complete GOase gene (bases 11917) by error-prone PCR (generations A1 and A2) and StEP recombination of improved variants from library A2 (generation A3) and (B) sequential random mutagenesis of a region of the GOase gene (bases 5181917) by error-prone PCR (generations B1B4).
Random mutagenesis and StEP recombination of the complete GOase gene. Error-prone PCR and StEP recombination (Zhao et al., 1998) were carried out using primers 5'-AATTCGAAGCTTATGGCCTCAGCACCTATCGGAAGC-3' (HindIII site underlined) and 5'-CCTCCTTCTAGATTACTGAGTAACGCGAATCGT-3' (XbaI site underlined). The mutagenic PCR reaction contained 10 mM TrisHCl, 50 mM KCl buffer (pH 8.5 at 25°C), ~0.3 µg plasmid DNA as template, 30 pmol of each primer, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP, 1 mM dTTP, 7 mM MgCl2, 0.1 mM MnCl2 and 1.5 U Taq polymerase (Perkin-Elmer, Gaithersburg, MD or Qiagen, Valencia, CA) in a total volume of 100 µl. PCR reactions were carried out on an MJ Research (Watertown, MA) thermal cycler (PTC-200) for 30 cycles with the following parameters: 94°C for 30 s, 50°C for 30 s and 72°C for 60 s. StEP recombination of four improved variants identified in generation A2 was performed in a 100 µl reaction containing 10 mM TrisHCl, 50 mM KCl buffer (pH 8.5), ~0.3 µg (total) plasmid DNA as template (prepared by mixing equal amounts of the four plasmids), 10 pmol of each primer, 0.5 mM of each dNTP, 2.5 mM MgCl2 and 5 U Taq polymerase. PCR conditions were as follows: 95°C for 3 min and 100 cycles of 94°C for 30 s and 58°C for 10 s. Mutagenic PCR or recombination products were purified using a DNA purification kit (Qiagen or Zymo Research, Orange, CA) and cloned (using the HindIII and XbaI restriction sites) back into the expression vector. Ligation mixtures were transformed into BL21(DE3) cells by electroporation.
Random mutagenesis of GOase gene region 5181917. Error-prone PCR was carried out using primers 5'-TTGTTCCTGCGGCTGCAGCAATTGAACCG-3' (PstI site underlined) and 5'-TGCCGGTCGACTCTCTAGATTACTGAGTAACG-3' (XbaI site underlined). Mutagenic PCR was performed in a 100 µl reaction mixture containing 10 mM TrisHCl, 50 mM KCl buffer (pH 8.3 at 25°C), 10 ng plasmid DNA as template, 50 pmol of each primer, 0.2 mM of each dNTP, 7 mM (generations B1 and B2) or 4 mM MgCl2 (generations B3 and B4) and 5 U Taq polymerase (Boehringer Mannheim). PCR conditions were as follows: 94°C for 2 min and 25 cycles of 94°C for 30 s, 58°C for 30 s and 72°C for 60 s. Purified restricted inserts from PCR reactions were ligated with an expression vector generated by PstIXbaI digestion of pGAO-036. Ligation products were transformed into BL21(DE3) cells by a modified chemical transformation method (SuperComp protocol, Bio 101, Inc., Carlsbad, CA).
Screening GOase libraries
Transformed cells were plated on LuriaBertani (LB) agar plates supplemented with 100 µg/ml ampicillin and grown overnight at 30°C.
Screening of libraries A1A3. Single colonies were picked into deep-well plates (well depth 2.4 cm, volume 1 ml; Beckton Dickinson Labware, Lincoln Park, NJ) and cells were grown for 10 h at 30°C and 270 r.p.m. in 200 µl LB medium containing 100 µg/ml ampicillin (LB-Amp). The master plates were duplicated by transferring a 10 µl aliquot to a new deep-well plate containing 300 µl LB-Amp and 1 mM IPTG and grown for 12 h at 30°C and 250 r.p.m. The cultures were then centrifuged for 10 min at 5000 r.p.m. and the cell pellet was resuspended in 300 µl 100 mM sodium phosphate (NaPi) buffer, pH 7.0, containing 0.4 mM CuSO4. Following addition of 0.5 mg/ml lysozyme (35 min at 37°C) and 2.5% (w/v) SDS (overnight at 4°C), the GOase activity was assayed using a GOase-HRP coupled assay (Baron et al., 1994). Aliquots of the cell extracts were reacted with D-galactose (50 mM for generation A1 or 25 mM for generations A2 and A3) at pH 7.0. The initial rate of H2O2 formation was followed by monitoring the HRP-catalyzed oxidation of ABTS at 405 nm on a Thermomax microplate reader (Molecular Devices, Sunnyvale, CA). Thermostability was assayed as follows: aliquots of the cell extracts were heated at 5570°C for 10 min and then chilled on ice for 10 min. The samples were equilibrated to room temperature, at which point they were assayed for activity. The ratio of residual to initial activity was used to characterize thermostability. 15002000 clones from each library were screened and the clones with improved activity or enhanced thermostability accompanied with activity comparable to the parents were picked for further verification.
Screening of libraries B1B4. Single colonies were picked into deep-well plates (well depth 4.4 cm, volume 2.2 ml; Qiagen) and cells were grown for 8 h at 30°C and 270 r.p.m. in 500 µl LB-Amp. The master plates were duplicated by transferring a 10 µl aliquot to a new deep-well plate containing 500 µl LB-Amp and 1mM IPTG and grown overnight at 30°C and 270 r.p.m. An aliquot of the culture was transferred to a microtiter plate. Following addition of 0.5 mg/ml lysozyme (30 min at 37°C) and 0.4% (w/v) SDS0.4 mM CuSO4 in 100 mM NaPi buffer, pH 7.0 (4 h at 4°C), the GOase activity was assayed using the GOaseHRP coupled assay as described above. The galactose concentration was 25 mM (generations B1 and B2) or 10 mM (generations B3 and B4). Approximately 1000 clones from each library were screened.
Protein purification and characterization
Escherichia coli cultures were grown for 16 h at 30°C in LB medium with 100 µg/ml ampicillin. Cells were harvested by centrifugation, resuspended in 100 mM sodium phosphate buffer, pH 7.0, containing 0.1 mM EDTA and disrupted by sonication. Cell debris was removed by centrifugation and the resulting supernatant was made 0.4 mM in CuSO4 and stirred for ~2 h at 4°C. (NH4)2SO4 was added to 25% saturation (w/v) and after centrifugation the supernatant was further saturated to 65% of (NH4)2SO4. The pellet was dissolved in 100 mM CH3COONH4 buffer, pH 7.2, and chromatographed on Sepharose 6B (Amersham Pharmacia, Piscataway, NJ) according to published procedures (Hatton and Regoeczi, 1982). Fractions with the highest GOase activity were collected and precipitated by addition of (NH4)2SO4 to 95% saturation. The pellet was dissolved in 100 mM NaPi, pH 7.0, and dialyzed extensively against the same buffer (the first dialysis buffer contained 0.2 mM CuSO4). The dialyzed protein was filtered through a 0.2 µm filter and frozen immediately at 80°C. Fungal GOase was purified by passage through a Sepharose 6B column. The eluted protein was incubated with 0.1 mM CuSO4 for 4 h at 4°C and desalted using a Biogel column (Bio-Rad, Hercules, CA).
The purified protein ran as a single band during SDSPAGE (Novex, San Diego, CA). Protein concentrations were determined from the absorbance at 280 nm ( = 1.05x105 M1 cm1) (Ettinger, 1974
). Kinetic measurements were performed in 100 mM NaPi buffer, pH 7.0, over a range of D-galactose concentrations from 15 to 250 mM using the HRPABTS coupled assay (Baron et al., 1994
). The rate of absorbance change was monitored by a Shimadzu (Columbia, MD) UV-Vis spectrophotometer at 420 nm (
= 42.3x 103 M1 cm1) (Yamazaki et al., 1997
). A LineweaverBurk plot was applied, and the kinetic parameters were calculated using the program KinetAsyst (IntelliKinetics, State College, PA).
UV-Vis spectra of wild-type and mutant GOases were recorded from 320 to 900 nm. Oxidation of GOase was performed in 100 mM NaPi, pH 7.0, by incubation with 100 mM K3[Fe(CN)6] for 10 min followed by removal of the oxidant on a Biogel column at 4°C (Whittaker and Whittaker, 1988).
Thermostability of the purified native, wild-type and mutant GOases was assessed by measuring residual activity/initial activity over the temperature range 2475°C. Enzymes (30 µl each, 0.054 mg/ml) were incubated at each temperature for 10 min in an MJ Research thermal cycler and chilled on ice before they were assayed in a microtiter plate. Stability at ambient temperature was measured by incubating 0.04 mg/ml GOase in the presence of 0.5 mM CuSO4 and 3 U of catalase in a total volume of 100 µl and measuring activity as a function of time.
![]() |
Results and discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The GOase gene from pR3 was amplified to introduce the ATG initiation codon as well as the HindIII and XbaI restriction sites, and the amplified fragment was subcloned into a pUC18 vector. Introduction of a second lac promoter led to increased expression (data not shown) and yielded plasmid pGAO-036. The cloned gaoA gene includes a pro-sequence encoding 41 amino acid residues, the N-terminal part of which has been proposed to be associated with secretion (McPherson et al., 1993) and which is cleaved in a copper-mediated self-processing reaction (Rogers et al., 2000
). Functional expression of low levels of mature GOase in E.coli was accomplished in the absence of the pro-sequence.
To increase the total activity of GOase in E.coli, random mutagenesis was applied to the entire mature GOase gene and also to just the region of the gene encoding domains II and III which are responsible for catalytic activity (McPherson et al., 1993). Adjusting the concentration of Mn2+ or Mg2+ during the PCR led to an error rate of ~23 base substitutions per gene. Mutant libraries were screened for activity on D-galactose, using HRP to detect the hydrogen peroxide produced during the reaction (Baron et al., 1994
). Higher-activity mutants identified in each round were subjected to further mutagenesis or recombination.
Screening 1600 mutants in the first round of mutagenesis of the complete GOase gene (library A1) generated two variants with 6- and 1.5-fold higher total activity toward D-galactose than the wild type (Table I). The most active variant A1.D12 showed enhanced thermostability as well and was used as template for the second generation. Screening ~1600 clones in the second generation identified four variants with improved activity and enhanced thermostability (A2.C3). Recombination of these four generated A3.E7 with ~60-fold higher total activity than wild-type GOase.
|
Protein purification, kinetics and spectroscopy
A rapid two-step procedure consisting of fractionation by (NH4)2SO4 and chromatography on Sepharose 6B was developed for purification of GOase. Recombinant wild-type GOase and mutants B4.F12 and A3.E7 were purified and characterized. All three migrated on SDSPAGE with an apparent molecular mass of 66 kDa rather than the 68.5 kDa predicted by the DNA sequence. The faster migration rate indicates that the thioether bond between Cys228 and Tyr272 is formed in all enzyme samples (Baron et al., 1994; Rogers et al., 2000
), in contrast to results with an earlier GOase E.coli expression system in which the majority of the enzyme produced was inactive because it lacked the thioether bond (McPherson et al., 1993
).
Kinetic parameters for fungal GOase and E.coli-expressed wild-type and mutant GOases are reported in Table II, as are the yields of purified enzymes. The fungal and wild-type recombinant GOases exhibit similar kinetic behavior. Variant B4.F12 shows an 8-fold increase in production of GOase at shake-flask level while retaining the catalytic efficiency of the wild type. The 30-fold increase in total activity for variant A3.E7 relative to wild-type reflects an 18-fold increase in GOase expression and a 1.7-fold increase in catalytic efficiency. This variant yields 10.8 mg/l purified enzyme.
|
Solutions of pure GOase as isolated are a mixture of two oxidation states, oxidized (Cu2+-Tyro) and semireduced (Cu2+-Tyr) (Whittaker and Whittaker, 1988). These states may be interconverted by treatment with oxidizing or reducing agents and are distinguished by their absorption spectra: the semireduced form shows weak absorption over the visible region whereas the oxidized form is characterized by an intense absorption band at 445 nm and a broad band with a maximum near 810 nm (Whittaker and Whittaker, 1988
; Baron et al., 1994
). Treatment of wild-type and evolved GOases with K3[Fe(CN)6] at 4°C led to the generation of the tyrosyl radical and the spectrum of the oxidized species was found to persist for several hours at ambient temperature (Figure 2
) indicating that the oxidized form of the enzyme produced in E.coli has redox potential and stability comparable to the GOase from Fusarium (Baron et al., 1994
; Reynolds et al., 1997
).
|
The thermostabilities of the purified fungal and E.coli-expressed wild-type and mutant GOases are shown in Figure 3. The T50 (the temperature at which the enzyme loses 50% of its activity following incubation for 10 min) is increased to 67°C for both B4.F12 and A3.E7 relative to a T50 of 63°C for wild type. However, the E.coli-expressed wild type is less stable than the fungal enzyme, whose T50 is 67°C. Similar results were obtained in the presence of 0.5 mM CuSO4 (data not shown). Possible reasons for the reduced thermostability of the recombinant wild-type enzyme may be that the disulfide bonds do not form properly, or may be related to the lack of glycosylation.
|
|
The mutations identified in the most highly expressed GOase variants are listed in Table I. Amino acid substitutions S10P, M70V and N413D as well as the synonymous mutations in codons S550 and S610 contribute to enhanced expression. The V494A substitution leads to increases in expression (variant B3.H7) and thermostability (variant A1.D12). G195E is a thermostabilizing mutation that appeared in clone A2.C3. The advantageous effect of N535D on expression became evident when recombined with other mutations in generation A3.
Identifying the mechanisms for the increased expression is difficult. However, we can provide some speculation. The mutation leading to amino acid substitution S10P is located at the N-terminal of the GOase gene; the nucleotide sequence in this region strongly influences gene transcription (Boer and Hui, 1990). Mutations could also contribute to changes in the secondary structure of GOase mRNA, which can affect protein expression (Cheong and Oriel, 2000
). Synonymous mutations S550 and S610 generate codons that are much less frequently used (TCT, 17.4 per 1000 to TCA, 1.0 per 1000). Using rare codons in specific regions of the gene can be advantageous for protein expression (Komar et al., 1999
), probably by inducing pauses in translation which result in a slower rate of protein synthesis and decreased levels of protein misfolding.
Screening the library made by recombining mutants A3.E7 and B4.F12 did not identify a more active variant than A3.E7. Nor did introduction of the N413D substitution into mutant A3.E7 by site-directed mutagenesis lead to a more active clone, demonstrating that the effects of the mutations are not cumulative.
Figure 5 shows the positions of the amino acid substitutions in variants B4.F12 and A3.E7. The V494A substitution is adjacent to the Cu(II) ligands Tyr495 and His496. The thermostabilizing mutation G195E occurs in a loop at the active site entrance and 10 Å away from copper. In wild type, Gly195 forms a hydrogen bond (2.90 Å) with Tyr189. A second charged hydrogen bond to Gly196 (2.40 Å) is introduced upon replacement of Gly with Glu, which may explain the beneficial influence of this mutation on GOase stability.
|
![]() |
Notes |
---|
2 Present address: Kyowa Hakko Kogyo Co. Ltd, 11 Kyowa-chou, Hofu-shi, Yamaguchi 747-8522, Japan
3 To whom correspondence should be addressed. E-mail: frances{at}cheme.caltech.edu
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Arnold,F.H. (1998) Acc. Chem. Res., 31, 125131.[ISI]
Avigad,G. (1985) Arch. Biochem. Biophys., 239, 531537.[ISI][Medline]
Avigad,G., Amaral,D., Asensio,C. and Horecker,B.L. (1962) J. Biol. Chem, 237, 27362743.
Baron,A.J., Stevens,C., Wilmot,C., Seneviratne,K.D., Blakeley,V., Dooley,D.M., Phillips,S.E.V., Knowles,P.F. and McPherson,M.J. (1994) J. Biol. Chem., 269, 2509525105.
Boer,H.D. and Hui,A.S. (1990) Methods Enzymol., 185, 103114.[Medline]
Bretting,H. and Jacobs,G. (1987) Biochim. Biophys. Acta, 913, 342348.[ISI][Medline]
Calderhead,D.M. and Lienhard,G.E. (1988) J. Biol. Chem., 263, 1217112174.
Cheong,T.K. and Oriel,P.J. (2000) Enzyme Microb. Technol., 26, 152158.[ISI][Medline]
Cooper,J.A.D., Smithe,W., Bacila,M. and Medina,H. (1959) J. Biol. Chem., 234, 445448.
Ettinger,M.J. (1974) Biochemistry, 13, 12421251.[ISI][Medline]
Gahmberg,C.G. and Tolvanen,M. (1994) Methods Enzymol., 230, 3244.[ISI][Medline]
Hatton,M. and Regoeczi,E. (1982) Methods Enzymol., 89, 172176.[ISI]
Ito,N., Phillips,S.E.V., Stevens,C., Ogel,Z.B., McPherson,M.J., Keen,J.N., Yadav,K.D.S. and Knowles,P.F. (1991) Nature, 350, 8790.[ISI][Medline]
Ito,N., Phillips,S.E.V., Yadav,K.D.S. and Knowles,P.F. (1994) J. Mol. Biol., 238, 794814.[ISI][Medline]
Komar,A.A., Lesnik,T. and Reiss,C. (1999) FEBS Lett., 462, 387391.[ISI][Medline]
Lis,M. and Kuramitsu,H.K. (1997) Antimicrob. Agents Chemother., 41, 9991003.[Abstract]
Liu,X.C. and Dordick,J.S. (1999) J. Am. Chem. Soc., 121, 466467.[ISI]
Mannino,S., Cosio,M.S. and Buratti,S. (1999) Ital. J. Food Sci., 11, 5765.[ISI]
Mazur,A.W. (1991) ACS Symp. Ser., 466, 99110.[ISI]
Mazur,A.W. and Hiler,G.D. (1997) J. Org. Chem., 62, 44714475.[ISI][Medline]
McPherson,M.J., Ogel,Z.B., Stevens,C., Yadav,K.D.S., Keen,J.N. and Knowles,P.F. (1992) J. Biol. Chem., 267, 81468152.
McPherson,M.J., Stevens,C., Baron,A.J., Ogel,Z.B., Seneviratne,K., Wilmot,C., Ito,N., Brocklebank,I., Phillips,S.E.V. and Knowles,P.F. (1993) Biochem. Soc. Trans., 21, 752756.[ISI][Medline]
Mendonca,M.H. and Zancan,G.T. (1987) Arch. Biochem. Biophys., 252, 507514.[ISI][Medline]
Petrounia,I.P. and Arnold,F.H. (2000) Curr. Opin. Biotechnol., 11, 325330.[ISI][Medline]
Reynolds,M.P., Baron,A.J., Wilmot,C.M., Vinesombe,E., Stevens,C., Phillips,S.E.V., Knowles,P.F. and McPherson,M.J. (1997) J. Biol. Inorg. Chem, 2, 327335.[ISI]
Rogers,M.S., Baron,A.J., McPherson,M.J., Knowles,P.F. and Dooley,D.M. (2000) J. Am. Chem. Soc., 122, 990991.[ISI]
Root,R.L., Durrwachter,J.R. and Wong,C.H. (1985) J. Am. Chem. Soc., 107, 29972999.[ISI]
Said,I.T., Shamsuddin,A.M., Sherief,M.A., Taleb,S.G., Aref,W.F. and Kumar,D. (1999) Histol. Histopathol., 14, 351357.[ISI][Medline]
Schlegel,R.A., Gerbeck,C.M. and Montgomery,R. (1968) Carbohydr. Res., 7, 193199.[ISI]
Szabo,E.E., Adanyi,N. and Varadi,M. (1996) Biosens. Bioelectron., 11, 10511058.[ISI][Medline]
Tkac,J., Gemeiner,P. and Sturdik,E. (1999) Biotechnol. Tech., 13, 931936.[ISI]
Vega,F.A., Nunez,C.G., Weigel,B., Hitzmann,B. and Ricci,J.C.D. (1998) Anal. Chim. Acta, 373, 5762.[ISI]
Vrbova,E., Peckova,J. and Marek,M. (1992) Collect. Czech. Chem. Commun., 57, 22872294.[ISI]
Whittaker,M. and Whittaker,J. (2000) Protein Express Purif., 20, 105111.[ISI][Medline]
Whittaker,M., Ballou,D. and Whittaker,J. (1998) Biochemistry, 37, 84268436.[ISI][Medline]
Whittaker,M.M. and Whittaker,J.W. (1988) J. Biol. Chem., 263, 60746080.
Whittaker,M.M., Devito,V.L., Asher,S.A. and Whittaker,J.W. (1989) J. Biol. Chem., 264, 71047106.
Xu,F., Golightly,E., Schneider,P., Berka,R., Brown,K., Johnstone,J., Baker,D., Fuglsang,C., Brown,S., Svendsen,A. and Klotz,A. (2000) Appl. Biochem. Biotechnol., 88, 2332.[ISI]
Yamazaki,T., Tsugawa,W. and Sode,K. (1997) Denki Kagaku, 65, 435439.[ISI]
Yang,G.Y. and Shamsuddin,A.M. (1996) Histol. Histopathol., 11, 801806.[ISI][Medline]
Zhao,H.M., Giver,L., Shao,Z.X., Affholter,J.A. and Arnold,F.H. (1998) Nature Biotechnol., 16, 258261.[ISI][Medline]
Received January 30, 2001; accepted May 15, 2001.