1 Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1, Yamada-oka, Suita Shi, Osaka, 565-0871, 2 The Chemo-Sero-Therapeutic Research Institute, Kawabe, Kyokushi, Kikuchi, Kumamoto 869-1298, 3 PRESTO, JST, 2-1 Yamadaoka, Suita, Osaka565-0871 and 4 Department of Pure and Applied Sciences, University of Tokyo, Komaba, Meguro-ku, Tokyo 153-8902, Japan
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Abstract |
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Keywords: esterase activity/phage display/protein evolution/random protein
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Introduction |
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Although nobody would ever provide the explicit route taken by natural proteins, experimental molecular evolution has imparted several insights towards the understanding of protein evolution (Matsuura et al., 1999; Arnold et al., 2001
; Kashiwagi et al., 2001
). We have previously shown that polypeptides with random sequences have a very slim chance of having such unique properties (Prijambada et al., 1996
; Yamauchi et al., 1998
). Recently, Keefe and Szostak showed roughly one in 1011 random polypeptides containing 80 contiguous random amino acids had ATP-binding ability (Keefe and Szostak, 2001
). Consecutively, it is possible to take new functional proteins directly from a large library (approximately 1013) of random polypeptides (Keefe and Szostak, 2001
; Wilson et al., 2001
) and further improve a property by directed evolution (Keefe and Szostak, 2001
).
How many random sequences need to be searched to permit the observance of evolving polypeptides towards acquiring higher functions? Is it feasible to decipher evolution from observing only a few variants per generation out of the vast number of available sequences, especially at the primitive stage? To address these issues, we deliberately limit our evolutionary studies on populations of arbitrarily chosen mutant random polypeptides to a maximum of 10 for each generation, although a phage display system with selection using transition state analogs (TSAs) has the capability of yielding highly diversified mutants of 1089 rendering it an efficient tool for generating a protein catalyst (Patten et al., 1996; Fujii et al., 1998
; Forrer et al., 1999
). Here, we show that even examining only 10 arbitrarily chosen polypeptides with random sequences per generation could guarantee the observance of evolution, in which there is a continuous gradual increase in a function of the polypeptides via iterating mutation and selection.
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Materials and methods |
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Escherichia coli strains used in this study were TG1 [K12, (lac-pro) supE thi hsdD5, F' traD36 proAB lacIq lacZ
M15] (Maniatis et al., 1982
) and BL21(DE3) (Novagen). Three phage clones displaying random polypeptides RP3-04, RP3-42 and RP3-45 were prepared as described previously (Nakashima et al., 2000
). Phage library PL1 and phagemid pCANSS were made available previously (Nakashima et al., 2000
). Helper phage M13KO7 was purchased from Amersham Pharmacia Biotech. PET21aSH vector for expressing C-terminal His6-tagged random polypeptides is a derivative of pET21a(+) (Novagen), in which the HindIII/XhoI cloning site was replaced with a HindIII/XhoI dsDNA fragment containing an SfiI site. The HindIII/XhoI dsDNA fragment was prepared by annealing 5'-AGCTTGGCCTCTGGGGCCGCACACCACCACCACC ACCACTAAC-3' with 5'-TCGAGTTAGTGGTGGTGGTGGTGGTGTGCGGCCCCAGAGGCCA-3'. The TSA, p-nitro-phenyl hydrogen 4-(hydroxycarbonyl)butylphosphonate (Ohkubo et al., 1994
), was kindly synthesized by Dojindo (Kumamoto, Japan).
Preparation of phage particles
In the case of the 10 random polypeptides per generation, each arbitrarily chosen phagemid clone harboring a variant gene of the polypeptide was grown in 2x YT (Maniatis et al., 1982) containing 100 µg/ml ampicillin and 2% glucose to an OD600 of 0.71.0, and the phage was rescued by adding the helper phage M13KO7 [2x1010 plaque-forming units (pfu)/ml]. After standing for 30 min at 30°C and shaking at the same temperature for another 30 min, the bacterial culture containing phage particles was centrifuged. The pellets rinsed with 2xYT containing 100 µg/ml ampicillin and 50 µg/ml kanamycin were suspended in the same medium and incubated at 30°C for 18 h. The cells were removed by centrifugation at 4°C for 15 min and the supernatant filtered by a Dismic-25 cs disposable syringe unit (0.45 µm, ADVANTEC MFS). The phage particles in the filtrate were precipitated by incubating at 4°C overnight after the addition of one fifth volume of 20% poly(ethylene glycol) 6000 (Wako Pure Chemical Industries) in 2.5 M NaCl. The phage particles collected by centrifugation at 10000 g for 20 min were resuspended in 50 mM TrisHCl (pH 7.0) containing 150 mM NaCl. The phage suspension was adjusted to a titer of 1013 transducing units (t.u.)/ml as estimated by the number of ampicillin resistant colonies (Marks et al., 1991
).
For the initial library of arbitrarily chosen 1000 clones, the preparation of phage particles is essentially the same as described above, except for the preparation of the library, from which the clones were derived. The library was prepared by consecutive error-prone and standard polymerase chain reaction (PCR) of the random polypeptide gene of the clone with highest binding affinity in the first generation of the 10-membered library. The mixture of the variant mutant genes so obtained was used to transform E.coli TG1, and from the transformants, 1000 clones were arbitrarily chosen, mixed, and used as initial library for the preparation of phage particles.
In vitro selection system
Each of the random polypeptides displayed on the surface of M13 phage as fusion proteins with the pIII coat protein were panned with the TSA (Figure 1a) of an esterase reaction (Ohkubo et al., 1994
). The TSA plate was prepared as follows: The 96-well NH2 microtiter plates (CovaLinkTM NH2 Module, NalgeNunc International) were coated with the TSA using the carbodiimide coupling method by the addition of 50 µl of 33 µM TSA in 0.1 M MES (pH 4.7) containing 0.15 M NaCl and 50 µl of 26 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). The plates were incubated overnight at 20°C before the plate was washed with 0.3 ml of water three times and blocked with glycine by incubating the plate for 1.5 h at 20°C after the addition of 0.3 ml of 1 M glycine. The well was then washed with 0.3 ml of 50 mM TrisHCl (pH 7.0) containing 150 mM NaCl three times before ready for use. Non-TSA plates were prepared with the same protocol except that 33 µM glycine was coated onto the wells of the microtiter plates instead of 33 µM TSA.
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Basically, the same selection system described above was applied to phage particles prepared from an initial mixture of 1000 clones. Phage particles were eluted after each of the 12 panning rounds, and the eluted mixed phages were used to infect E.coli TG1. All transfectants of a generation were collected and used for the preparation of phage particles.
Random mutagenesis and construction of the phage library
A random polypeptide gene on the phagemid, prepared from E.coli TG1 cells infected with a selected phage, was amplified by error-prone PCR as described previously (Arakawa et al., 1996) using the Thermus thermophilus DNA polymerase and the primers, 5'-CCGCCTTTGAGTGAGCTGAT-3' and 5'-TCGTCACCAGTACAAACCACAACG-3'. The mutated products were isolated and further amplified by standard PCR as described previously (Nakashima et al., 2000
). For the first to fourth generations, the forward primer used for standard PCR was 5'-ATCCTCGCAACTGCGGCCCACGTGGCCAT-GGCTAGCATGACTGGTGGACAGCAAATGGGT-3' and the reverse primer was 5'-AGTTTAGGCCCCAGAGGCTAATCGCAGTCTGTCGACTC-3' to allow screening for the BglI restriction site. For the fifth and sixth generations, primers used were basically the same except that the 19th base of the reverse primer was changed to C instead of T to generate SfiI restriction site for screening purposes. The change of base on the primer did not affect the amino acid sequences. The BglI or SfiI fragment from the PCR products was ligated into pCANSS, and E.coli TG1 cells were transfected with the ligated DNA as described previously (Nakashima et al., 2000
). The phage library was prepared from the phagemid library as described previously (Nakashima et al., 2000
).
Expression and purification of His-tagged random polypeptides
A polypeptide gene on the phagemid carried by a selected phage was recloned into the NheI/SfiI site of the newly constructed expression vector, pET21aSH, described above. The E.coli BL21(DE3) cells harboring the hybrid plasmid grown at 37°C on LB medium (Maniatis et al., 1982) containing 50 µg/ml ampicillin to an OD600 of 0.61.0 was further incubated for 5 h at 30°C after the addition of isopropyl-ß-D-thiogalactopyranoside (IPTG) (final concentration, 1 mM). Cells harvested after IPTG induction were disrupted in 50 mM potassium phosphate (pH 8.0) containing 8 M urea, 0.3 M NaCl and 10 mM imidazole, and the cell suspension centrifuged. The supernatant was applied to a Ni-NTA Superflow (Qiagen) column equilibrated with the same buffer used for disrupting the cells. The column was washed with 50 mM potassium phosphate (pH 8.0) containing 8 M urea, 0.3 M NaCl and 20 mM imidazole, and the polypeptide was eluted with an imidazole gradient of 2080 mM. The fractions showing a single band on SDSpolyacrylamide gel electrophoresis (SDSPAGE) were pooled, and the pooled fractions transferred to Spectra/Por® dialyzing membrane (MWCO: 3500; Spectrum Laboratories) and concentrated by surrounding the tube with poly(ethylene glycol) 20 000 (Wako Pure Chemical Industries). After incubating at room temperature for 6 h, the tube was gently rinsed with distilled water and dialyzed against 62.5 mM sodium acetate, pH 5.0. Dialysis was first carried out at room temperature with two buffer changes, each at 2-h intervals, to completely remove the urea from the polypeptide solution. The polypeptide solution was further dialyzed at 4°C with three buffer changes at consecutive 2-, 12- and 1-h intervals. The dialyzate was sterilized using a Dismic-25 cs disposable syringe unit (0.45 µm) and the purified polypeptide was stored at 4°C.
All purified polypeptides were confirmed homogeneous by SDSPAGE, and the amino acid compositions coincided well with those deduced from the nucleotide sequences. The molar absorption coefficients of the polypeptides at 280 nm, determined from the amino acid composition and A280 value of the purified polypeptide solution as previously described (Suga et al., 1996), were as follows: RP3-42H, 37000 M1 cm1; YSLP1-1, 63000 M1 cm1; YSLP3-1, 67000 M1 cm1; and YSLP6-1, 65000 M1 cm1.
Esterase activity
The hydrolysis rate of p-nitrophenyl acetate was measured at 37°C. Reactions were initiated by mixing 20 µl of 2.5 mM p-nitrophenyl acetate in 1.25% methanol with 80 µl of a polypeptide solution containing 62.5 mM sodium acetate (pH 5.0). After incubating for 1, 2, 3 or 4 h, the reaction mixture was chilled on ice and the pH was increased to 7.2 by adding 25 µl of 250 mM potassium phosphate (pH 8.0). The precipitate formed after the pH shift of the reaction mixture was separated by centrifugation, and the absorbance of the supernatant at 400 nm was measured. The 0 h sample was prepared in the same manner, except that the substrate and polypeptide were mixed on ice without incubation at 37°C. To examine the effect of the TSA, different concentrations of the TSA (0.47, 2.35 and 4.65 mM) were added into the substrate solution used for the reaction. The concentration of p-nitrophenol released by the reaction was calculated using a molar absorption coefficient of 10400 M1 cm1, which was determined experimentally at pH 7.2.
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Results and discussion |
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The first generation comprised of RP3-04-, RP3-42- and RP3-45-displaying phage (Nakashima et al., 2000) and seven clones displaying different random polypeptides arbitrarily chosen from a large library (PL1) constructed previously (Nakashima, et al., 2000
). RP3-04, RP3-42 and RP3-45 are soluble random polypeptides with no secondary structure propensity but are as compact as a molten globule (Yamauchi et al., 1998
). In addition, all the displayed random polypeptides have no homology with known natural proteins in the SwissProt database as analyzed by BLAST 2.2.1 (Figure 2a
). Hence, the initial material of the evolutionary study could be assumed to be like any primitive sequence in view of evolvability regarding affinity to the TSA. When the 10 phage clones of the first generation were assayed for binding affinity, significant differences in binding ability to the TSA were observed (Figure 1b
), implying that even from a small library of 10 random polypeptides, experimentally detectable variety could be found. Hence, sampling of as few as 10 from a huge number of possible sequences permits the observation of an evolving population.
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The deduced amino acid sequences of the polypeptide region of all the members in each generation are listed in Figure 2a, and no homology was detected between the selected random polypeptides and the natural proteins with known amino acid sequences. As shown in Figure 2a
, even with only a few mutations accumulated, 13 sites, from the initial stage to the sixth generation, the acquired binding capacity of the mutant YSLP6-1 in the sixth generation is significantly high as compared to those in the initial stage of the experimental evolution. The easy ascent towards acquiring a new property, the binding affinity, by a small number of mutations in the random polypeptides, indicates that a significant fraction of amino acid sequences other than the known natural proteins have a certain function, at least in the binding activity.
Will an increase in variety make any difference in the observation of protein evolution? About 1000 clones were arbitrarily chosen from the mutant polypeptides generated from the selected clone in the first generation, and were subjected to several panning rounds with the TSA. The binding affinity of the clones increased gradually on each panning round but becomes saturated on the 11th round, as shown by no further increase in the binding affinity of the population (Figure 3). Out of the 10 clones sampled from the final population after the panning rounds, nine were found to have the same amino acid sequence (two amino acid substitutions, W46R and L75P, from the parent sequence of the second generation) and one has the same sequence as that of the third clone in the second generation (see Figure 2b
). In addition, the binding affinity of the major clone (4.2x103 t.u.) was found to be similar to the clone with highest binding affinity (3.8x103 t.u.) in the second generation. These results infer that a large variety is not always effective for obtaining a good mutant.
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Considering that the TSA is a transition state analog for an esterase reaction, the esterase activity of the polypeptides may have increased through the process of evolution, as a number of studies have demonstrated that catalytic efficiency usually increases as a function of increased affinity for a respective TSA (Philipps et al., 1992; Angeles et al., 1993; Stewart and Benkovic, 1995
). Note that the binding affinity of the random polypeptides was estimated by the titer of the M13 phage displaying the polypeptide as a fusion protein with the pIII coat protein. Therefore, to assess the kinetic parameters for the esterase activities of the selected (advantageous) polypeptides in different generations accurately, the polypeptides were detached from the fused protein displayed on the phage prior to purification. The polypeptide genes in the phagemids from RP3-42-displaying phage clones, the clones showing the highest binding affinity toward TSA in the first, third, and sixth generations, were independently recloned to an expression vector as described in Materials and methods. The expressed polypeptides with a His6 tag were then purified and named RP3-42H, YSLP1-1, YSLP3-1 and YSLP6-1, respectively. The deduced amino acid sequences are shown in Figure 2b
. The esterase activity of the purified polypeptides was measured using p-nitrophenyl acetate as a substrate (Figure 4
). In addition, it was examined whether the TSA inhibits the activity competitively.
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![]() | (1) |
where KI is the inhibition constant, kB is the rate constant for ester hydrolysis in the absence of a polypeptide, E, i.e. the rate constant of the background reaction. Here, KI is equivalent to the ratio of the dissociation rate constant to the association rate constant, and hence KI represents the dissociation constant, Kd, of TSA. When the activity of a polypeptide is at a primitive level, the Michaelis constant, Km, must be much larger than the mM order. Hence, applying this to our experimental conditions where the initial concentration of ester, [S]0, is 0.5 mM, (1 + [I] / KI)Km >> [S]. In such a case, Equation (1) is simplified to
![]() | (2) |
Integration of equation (2) gives:
![]() | (3) |
where t is the reaction time, and [P]t is the concentration of p-nitrophenol released by the reaction at time t. In the absence of the inhibitor, Equation (3) is simplified to
![]() | (4) |
Figure 4 shows that ester hydrolysis catalyzed by the polypeptides followed a linear relationship expected from Equation (4), in agreement with large Km values. Figure 5
shows the effect of polypeptide concentration on the slope of the linear lines for the esterase hydrolysis, as shown in Figure 4
. The linear lines for RP3-42H, YSLP1-1, YSLP3-1 and YSLP6-1 intersect at the y-axis. The intersecting points show a kB value of 2.3x106 s1 for the background reaction and the slope of each line gives kcat / Km values of 0.16, 0.49, 0.75 and 1.07 M1 s1 for RP3-42H, YSLP1-1, YSLP3-1 and YSLP6-1, respectively (Table I
). These results clearly show that the polypeptides are evolving towards acquiring higher catalytic functions. A negative control (Yamauchi et al., 1998
) ruled out the possibility that the observed esterase activity is due to contamination of enzyme(s) present in the host cell.
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Given that transition-state analogs serve as respective inhibitors to enzyme-catalyzed reactions and that catalytic efficiency is a function of binding affinity (Philipps et al., 1992; Angeles et al., 1993), inhibition of esterase activity by the addition of TSA was investigated. While TSA showed no influence on the activity of RP3-42H, an increase in inhibitory effect was observed towards reactions catalyzed by YSLP6-1 upon an increase in the concentration of TSA (Figure 6a
). In addition, it was confirmed that the TSA addition does not affect the background reaction (Figure 6a
). Applying equation (3
) to the slopes of YSLP6-1 in Figure 6a
, [E] / (slope kB) which corresponds to {(1 + [I] / KI)Km} / kcat is plotted against the concentration of the inhibitor. From the slope of the linear line of YSLP6-1 in Figure 6b
, a I value was calculated using kcat / Km values in Table I
K. Furthermore, the inhibition constant KI, which corresponds to the dissociation constant, Kd, between the polypeptides and the TSA, decreased significantly on generations, guaranteeing that there is an increase in the esterase activity of the selected polypeptides over the generations even when the polypeptides were detached from the phage. In addition, these results implied that the affinity assay, by panning to the TSA as a selection system, is reliable.
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Notes |
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Acknowledgments |
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References |
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Received October 10, 2001; revised March 19, 2002; accepted March 23, 2002.