1 Department of Functional Materials Science, Saitama University,255 Shimo-Okubo, Saitama 338-8570 and 2 GenCom Co., 11 Minami-Oya, Machida 194-8511, Japan
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Abstract |
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Keywords: block shuffling/diversity/evolutionary protein engineering/GFP/library
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Introduction |
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Recent findings in molecular biology, especially in the field of genome science, have been establishing that proteins have evolved through recombinations such as domain shuffling (Doolittle, 1995), exon shuffling (Kolkman et al., 2001) and module shuffling (Roy et al., 1999
). In addition to these sophisticated shuffling mechanisms, recombination itself seems to have contributed to the molecular evolution in the form of general homologous/non-homologous mechanisms and transposable element-mediated mechanisms (Kornberg and Baker, 1992
; Fedoroff, 1999
). It is impressive that, with a relatively small number of genes (at most 40 000 genes) contained in the whole human genome, humans can generate a highly complex and sophisticated molecular system as a result of alternative splicing (Kondrashov and Koonin, 2001
; Li et al., 2001
). Consequently, these facts seem to support the idea that block-shuffling mechanisms can mine functional proteins effectively.
Protein engineering can be performed based on the two fundamental mutation technologies: substitution and recombination. The former has been well developed, including site-directed mutagenesis and chemical synthesis methods (Botstein and Shortle, 1985; Sambrook and Russell, 2001a
).
In contrast to the well developed substitution technology, the other important technology, recombination, is yet to be exploited. In general, recombination can be fulfilled by two types of technology: ligation by enzymes (Nishigaki et al., 1995; Sambrook and Russell, 2001b
) and homology-based PCR just as that used in DNA shuffling (Stemmer, 1994
). Both are already used routinely in combining a few DNA fragments (Wakasugi et al., 1997
; Tsuji et al., 1999
; Kikuchi et al., 2000
) or in random-assembling of DNA fragments as in the microgene method (Shiba et al., 1997
) and others (Shao et al.,1998
; Christians et al., 1999
; Riechmann and Winter, 2000
). These technologies usually require short stretches of DNA sequences as working sequences (such as recognition sequences of restriction enzymes or homologous sequences for generating priming structures for PCR), thus imposing a constraint on these sequences except in the case of a flush-end ligation (which is governed by chance and gives a very small yield).
In this work, we report a novel technology for block shuffling of DNA, and consequently, block shuffling of protein, based on Y-ligation [i.e. ligation of blocks with a stem and two branches (Nishigaki et al., 1999)], developing a new field for evolutionary protein engineering. This technology is demonstrated to be applicable to shuffling of blocks of various sizes (amino acid monomer to polypeptide size). The significance of block shuffling for evolutionary protein engineering is also discussed.
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Materials and methods |
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Two types of single-stranded DNAs (5'-half and 3'-half strands, see Figure 1) were prepared as shuffling device sequences. These DNAs contain a single block sequence that is situated either at the 3'- or 5'-end. The strands are made complementary at their stem region and contain a D-branch region as depicted in Figure 1a
, which works as a primer-binding site for PCR. The 5'-end of the 3'-half strand should be phosphorylated for a ligation reaction. Equal amounts of the 5'-half and the 3'-half strands (usually 10 pmol each in 10 µl) were combined and hybridized through their stem regions. The 3'-end of the 5'-half strand and the 5'-end of the 3'-half strand were ligated using 50 U T4 RNA ligase (Takara, Kyoto, Japan) in the presence of 0.1 mM ATP. After pre-amplification of the ligation products (though it is possible to omit this step), two types of PCR were performed to obtain pre-5'-half and pre-3'-half PCR products. In advance, the primers containing the stem sequence (primers pS5 and pS3 in Figure 1
) were biotinylated at the 5'-end in order to be used for preparing the ssDNAs for the next Y-ligation cycle. The PCR products were separately digested with the corresponding restriction enzyme (MboII for 3'-half and AlwI for 5'-half) and then ssDNAs were collected through avidin-biotin binding (streptavidin-coated magnetic beads were used). The biotinylated strands of the pre-5'-half and the non-biotinylated strands of the pre-3'-half (Figure 1, top
) were then used as the 5'-half strand and the 3'-half strand, respectively, in the next Y-ligation cycle. Depending on the cycle number, n, the size and the diversity of ligated blocks were allowed to increase exponentially: 2n (= s) for the size and ds for the product diversity where d is the number of block diversity at the start (note that the latter increases in a series of 64, 4096, 1.7x107, 2.8x1014 if d = 8).
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All oligonucleotides listed in Tables I and II were custom-synthesized [by either Nihon Bio Service (Asaka, Japan), Sawady Technology (Tokyo, Japan) or Amersham Bioscience (Tokyo, Japan)]. The starting materials for YLBS and non-labeled primers for PCR were of cartridge grade. Biotinylated and FITC-labeled primers used for PCR were of HPLC or PAGE grade. The starting materials used for the subtilisin library were prepared by PCR using oligonucleotides (Table II
).
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The 5'-half and the 3'-half DNAs in 10 µl of ligation buffer containing 50 mM TrisHCl (pH 8.0), 10 mM MgCl2, 10 mg/l BSA, 1 mM hexamminecobalt (III) chloride, and 25% polyethylene glycol 6000 (Tessier et al., 1986) were annealed through heating at 94°C (5 min) and then at 60°C (15 min). Ligation was then carried out with 50 U T4 RNA ligase (Takara) in the presence of 0.1 mM ATP at 25°C for 16 h.
Amplification of ligated products by PCR
Ligated products were purified by denaturing polyacrylamide gel electrophoresis (8 M urea, 8% acrylamide; 250 V for 45 min). Requisite bands were excised from the gel, washed with 1 ml of water and finally crushed in 50 µl of water using a gel-crushing rod. One microliter of this extract was used for PCR to confirm the success of ligation and led to the preparation of 5'-half and 3'-half strands. PCR was performed using 10 pmol of primers (see Table II) in 50 µl of a PCR buffer containing 200 µM of each deoxyribonucleotide triphosphate (dNTP), 1 U Taq polymerase (Greiner, Tokyo, Japan), 50 mM TrisHCl (pH 8.7) and 2.5 mM MgCl2. The cycle of pre-denaturation (90°C, 2 min), denaturation (90°C, 30 s), annealing (60°C, 1 min) and extension (72°C, 30 s) was repeated for 30 rounds. Additional steps of PCR and gel purification could be used to further purify the products.
Restriction digestion
The pre-5'-half and the pre-3'-half PCR products were prepared using the ligation products recovered by ethanol precipitation. Each DNA was incubated with 10 U restriction enzyme [MboII (Takara), AlwI (New England Biolabs, Beverly, USA) or MboI (Takara)] in 10 µl of a buffer {MboII buffer [10 mM TrisHCl (pH 7.5), 10 mM MgCl2 and 1 mM DTT], AlwI buffer [20 mM TrisHCl (pH 7.9), 10 mM MgCl2, 1 mM DTT and 50 mM KCl] and MboI buffer [20 mM TrisHCl (pH 8.5), 10 mM MgCl2, 1 mM DTT and 100 mM KCl]} at 37°C for 1 h. The surrounding sequences of the recognition sequences are shown in Figure 1.
Preparation of ssDNAs from PCR products (pre-5'-half and pre-3'-half)
To each restriction digest (10 µl) were added 40 µl of water and 50 µl of a streptavidin-coated magnetic bead [Dynabeads M-280 Streptavidin (Dynal, Oslo, Norway)] suspension. Beads were pre-treated with 0.1 M NaOH and equilibrated in 0.1 M TrisHCl (pH 8.0), 0.5 M NaCl and 1% Tween-20 prior to use. The DNA/bead suspension was shaken at room temperature for 1h. The collected beads were then washed three times with 100 µl of 0.01% BSA and treated twice with 25 µl of 25% (w/v) ammonium hydroxide at room temperature for 2 min to recover any non-biotinylated ssDNA (Jurinke et al., 1997). After further washing with 100 µl of 0.01% BSA, biotinylated ssDNA was recovered by treating the beads twice with 25 µl of 25% (w/v) ammonium hydroxide at 65°C for 15 min, subsequently dried in vacuo. The biotinylated ssDNA recovered from the pre-5'-half and the non-biotinylated ssDNA from the pre-3'-half were used as 5'-half strand and 3'-half strands for the next cycle, respectively.
Cloning and sequencing
The shuffled DNAs of the GFP gene fragments were digested with AatII and NspV and then cloned into a specified plasmid vector as described below. Shuffled DNAs for peptide and subtilisin libraries were cloned into plasmid pCR2.1 using a TA cloning kit (TA Cloning Kit Jr. or TOPO TA Cloning; Invitrogen, Carlsbad, CA, USA). Recombinant plasmids were transformed into Escherichia coli DH5 or TOP10 (Invitrogen) by electroporation using Gene Pulser (Bio-Rad, Richmond, VA, USA) or the calcium chloride method (Sambrook and Russell, 2001c
). The plasmid DNA was purified from 5 ml cultures using a plasmid extraction kit, Wizard Plus SV Minipreps DNA Purification System (Promega, Madison, WI, USA). DNA sequences were determined using a DNA sequencing kit, Thermo Sequenase fluorescently-labeled primer cycle sequencing kit with 7-deaza-dGTP (Amersham Pharmacia Biotech, Little Chalfont, WI, USA) and a DNA sequencer, DSQ2000 (Shimazu, Kyoto, Japan). In a few cases, custom-sequencing (Sawaday Technology) was adopted.
Vector preparation of the GFP library
Plasmid pGFPuv4-NF, which contains a non-fluorescent GFP gene coding sequence, was prepared by restriction digestion with NcoI (Takara) followed by treatment of pGFPuv4 (Ito et al., 1999) with T4 DNA polymerase (Takara) and re-ligation with T4 DNA ligase (Takara). The expression vector was prepared from plasmid pGFPuv4-NF by PCR using GFP-D1 and GFP-D2 (10 pmol each, Table II
) as primers. These primers contain AatII and NspV restriction sites and are designed so as not to modify the open reading frame of the GFP gene. PCR assays consisted of 30 cycles of denaturation (94°C, 30 s), annealing (55°C, 30 s) and extension (72°C, 2.5 min) after pre-denaturation (94°C, 2 min). The PCR products were finally treated with AatII (New England Biolabs) and NspV (Takara) and purified using a QIAquick column (QIAGEN, Hilden, Germany).
Selection of GFP library
Block-shuffling products of four blocks (CF) were attached with blocks A + B (at the upstream of the four blocks) and blocks G + H (downstream of them) through PCR procedures. For cloning into an expression vector (see above), the products were attached with linkers by PCR using primers, GFP-P1 and GFP-N1, which contain restriction sequences for AatII and NspV, respectively, to fit with the expression vector. Then, they were treated with the restriction enzymes AatII and NspV and incubated in a solution containing 4 fmol of expression vector and 4 Weiss units of T4 DNA ligase (Takara) at 16°C for 18 h. The ligation mixture was subjected to electroporation with 80 µl of a competent cell (E.coli strain DH5) suspension using an electropulser (Bio-Rad) under the conditions of 1.8 kV and 4 ms of pulse width. To these was added 2 ml of SOB medium. The samples were incubated at 37°C for 1 h, then plated in 1 ml aliquots onto an A4 size LB agar plate (210x280 mm) which contained 100 µg/ml ampicillin, and incubated at 37°C for 18 h. The culture plates were analyzed using a fluoroimager, Molecular Imager FX (Bio-Rad, Hercules, CA, USA) using wavelengths of 488 nm (excitation) and 515545 nm (emission).
PAGE and temperature-gradient gel electrophoresis (TGGE)
Denaturing gel electrophoresis (8 M urea, 8% acrylamide) was performed at 60°C (at 250 V for 45min). The running buffer contained 40 mM Tris-acetate (pH 8.0), 20 mM sodium acetate and 2 mM EDTA. To evaluate the diversity of shuffled DNAs, PCR products were analyzed using TGGE. Gels consisted of 8 M urea and 6% acrylamide (acrylamide: bis-acrylamide, 19:1) in 1x TBE buffer [90 mM Tris-borate and 2 mM EDTA (pH 8.0)]. TGGE was performed under a temperature gradient of 2070°C using Thermo Gradient TG (TAITEC, Saitama, Japan). DNA bands were detected with silver staining.
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Results and discussion |
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Products of each intermediate step and their diversity.
A serial procedure of YLBS (Figure 1) was devised and performed on GFP blocks (see Table I
) following the protocols described in Materials and methods. GFP was selected because of its fluorescent nature which is useful for screening; it was divided into eight blocks of 30 nucleotides (Table I
). The ligation products of GFP blocks at each step (Y1Y3) were obtained in a yield of approximately 550%, extracted from a gel and then PCR amplified (Figure 2
). [Note that the amount of the seed DNA for PCR extracted from a gel, which was estimated to be more than 1 fmol (
109 molecules), exceeds the whole diversity of Y3 (= 88 = 1.7x107) in this case.] A population of shuffled DNAs could be purified to an apparently single band in gel electrophoresis (except Y3 in this case, which needed another purification step) due to the same degree of polymerization. The diversity of the products at each step can be readily monitored by using TGGE as shown in Figure 3
. The complicated transition pattern indicates that the apparently single band consists of various DNA strands. From the range of the initial transition of these strands, we can make a rough estimation of the diversity of the shuffled DNAs as shown in Figure 3
. Namely, a possible DNA sequence which has the lowest or highest melting temperature among the shuffled DNAs was predicted, mainly based on G+C content, and then subjected to a prediction program for the DNA melting profile (Steger, 1994
) to obtain the theoretical transition temperatures. Though the values obtained are known to deviate from the true ones with a definite relationship (Nishigaki et al., 1984
, 2000
; Abrams and Stanton 1992
), the relative values are sufficiently reliable (Nishigaki et al., 1984
). The difference between the highest and the lowest melting temperatures,
T (=T2 T1), for a population of shuffled DNAs, could be theoretically obtained as 4.0°C, and was comparable with the experimental result (4.8°C) obtained with TGGE (Figure 3
). As theoretically expected, the transition profile of shuffled DNAs can be taken as a composite of many transitions occurring at various temperatures, providing a qualitative confirmation of diversity. Thus, TGGE is a convenient, though less accurate, way of diversity monitoring.
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Shuffling of amino acid monomer-sized blocks and others
There are intriguing points in dealing with a single amino acid (or a minimum-sized block of peptide) as a block since it can provide us with another way to synthesize, in theory, an arbitrary sequence of protein with a favorable occurrence of amino acids and without interference of stop codons. Therefore, it was examined whether we can shuffle trinucleotides (we call this aa-block here) by YLBS technology.
Shuffled products of aa-blocks.
Seven species of trinucleotides corresponding to Gly, Ile, Asp, Lys, Ser, Cys and Pro were chosen as starting blocks (Table I) by considering the codon usage of wheat germ (Ikemura, 1985
). These blocks were attached with the shuffling device sequence of 5'-half or 3'-half (see Figure 1
), which has a minor change in their sequence to adapt to the restriction enzyme employed (Table I
). Using the essentially identical procedures used in YLBS of GFP blocks (Figure 1
), aa-blocks were ligated to dimers (Y1), tetramers (Y2), octamers (Y3) and hexadecamers (Y4). The diversities of these libraries (Y1Y4) could be checked in the same way as shown in Figure 3
. The final products (Y4) were cloned and sequenced (68 clones) as partly shown in Figure 4d
. Ligation of 16 blocks after the fourth step was confirmed to be approximately 8% of the final products (thus, 4.8x1010 diversity), showing the capability of aa-block shuffling. However, there also happened to be a high frequency of deletion as in the previous experiments with GFP blocks. The same situation continued for four similar independent experiments (the statistics of these are shown in Table IV
). The occurrence of each aa-block was within a statistically permissible range of fluctuation. Therefore, we concluded that aa-blocks could be shuffled by YLBS technology. This technology is more powerful in generating a diversity of DNAs encoding polypeptides than the conventional method that synthesizes nucleotide by nucleotide since YLBS can synthesize DNAs by the unit of trinucleotides or hexanucleotides, and so on.
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Solution for deletion problems
The stubborn problem of deletion encountered in the shuffling of both module-sized blocks and aa-blocks has finally been solved by our independent experiments. The main causes for deletion phenomena were determined to be: (i) impurity of starting blocks and (ii) anomalous excision activity of type IIS restriction enzymes. From the close inspection of deletion products (partly shown in Figure 4), in which deletion was strongly associated with the second strand scission by type IIS restriction enzyme MboII, a hypothesis was presented that an anomalous cleavage occurs in the scission of the second strand, which follows after the cleavage of one strand, probably due to the instability of the nick-containing structure of the substrate DNA. Based on this hypothesis and assuming the strand to be cleaved secondly, we made up a new construct of a Y-ligation device sequence which does not depend on the abnormal second strand scission (Figure 6a
). Using this construct, we could build up YLBS libraries of <10% deletion (as partly shown in Figure 6b
), leading to the final solution for the deletion phenomena.
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On the other hand, the deviation problem is rather serious since it means that some poorly-distributed block species cannot make a substantial contribution to the formation of a shuffled library. In addition, the deviation may reflect the essential property of the molecular device employed, i.e. T4 RNA ligase, on the interaction with various substrate DNA sequences (e.g. the trinucleotide of CCC may be more favored as a substrate in the ligation reaction than that of GGG). This seems to be the case and, fortunately, is not so extreme, based on our experimental data (partly shown in this work). This leads us to take two types of measures against these problems: (i) try not to use nucleotide sequences which provide poor substrates for T4 RNA ligase and (ii) try to raise the yield of Y-ligation as much as possible since the yield of 100% stands for no deviation as all the molecules have been involved in the reaction. The former is more realistic and the latter is a desirable aim. Therefore, our big technological challenge can be said to be to increase the yield of Y-ligation.
Block-shuffled libraries essential for protein engineering
Block shuffling can generate a well defined, well distributed library of proteins, which is definitely important for protein engineering as stated below. As a result of the studies on evolutionary molecular engineering (Eigen and Gardiner, 1984; Voigt et al., 2000
; and other works), it is now evident that once a protein of a function is discovered (however weak its activity may be), then it can be evolved to provide a better function by hill-climbing through point mutations. In particular, it is known to be effective to accumulate advantageous point mutations by sexual PCR (Stemmer, 1994
) or the like. Unfortunately, there is no general rule established that enables us to find a de novo functional protein used for evolution. Here, the well defined and well distributed libraries generated by block shuffling can work as convenient initial materials to be examined. This is because the well defined library enables us to design the next library, of which members are the most different in sequence from those of the former library, allowing us to evade testing the same or close sequences which have already been tested before. Therefore, point mutation is a way to walk in a small stride whereas block shuffling is a way to walk in a wide stride with controlled walking. (Even point mutations can make a wide stride by virtue of their high frequency, but the resultant products are unpredictable and uncontrollable in general.)
The most significant benefit provided by this technology must be in the readiness to integrate a meaningful block, irrespective of its size [from amino acid monomer to domain (approximately 100 amino acids) or more], into proteins at any site at any frequency. Now we can examine what is the effect if we dope a particular peptide sequence at various locations of a protein or what will happen if a null block (or nothing block) is inserted (in other words, introduction of block deletion) at various sites of a protein. Meaningful blocks can be either exons, domains or modules. Obviously, YLBS is the first general technology that has enabled us to shuffle exons and domains at will, with ease, and in a huge scale of diversity, opening the gate wide for evolutionary protein engineering. This is especially important when modularity is becoming a more important key concept in evolution (Go, 1981; Dover, 2000
).
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Notes |
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K.Kitamura and Y.Kinoshita contributed equally to this work
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Acknowledgments |
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References |
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Received March 18, 2002; revised June 17, 2002; accepted July 2, 2002.