Department of Molecular Science and Technology, Ajou University, San5, Woncheon-dong, Paldal-gu, Suwon, 442-749 and 1 Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1, Kusung-dong, Yusung-gu, Taejon, 305-701, Korea
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Abstract |
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Abbreviations: FSS, functional salvage screen GFPuv, green fluorescent protein with an enhanced fluorescence MBP, maltose-binding protein GFP176(+1), a defective GFPuv constructed from the wild-type protein by deletion of 176V with an additional base GFP
176(+2), a defective GFPuv constructed by deletion of 176V with additional two bases GFP
1723/176(+1), a defective GFPuv constructed by deletion of two further residues 172E and 173D from GFP
176(+1) GFP
1723/176(+2), a defective GFPuv constructed by deletion of two further residues 172E and 173D from GFP
176(+2) GFP
129138/176(+2), a defective GFPuv constructed by deletion of the region 129D138G from GFP
176(+2).
Keywords: functional salvage screen/green fluorescent protein/new lineage/protein engineering/sequence space
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Introduction |
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Conventional mutagenesis and directed evolution techniques generally give rise to mutations on the target gene in a random fashion and a library of variants having the same sequence space as that of the parent gene is subjected to screening (Stemmer, 1994). Thus, most of the variants lie within the pre-existing and structurally fated sequence space, excluding the chances of creating protein lineages with new fitness landscapes. Recently, random elongation mutagenesis was attempted to generate the variants through the addition of random peptide tails to the C-terminus of the enzyme, providing a clue that changing the sequence space of a protein by incorporation of a random sequence could generate diverse protein lineages (Matsuura et al., 1999
). In line with this, a number of approaches including sub-domain swapping (Hopfner et al., 1998
; Kumar and Rao, 2000
), domain or module grafting (Greenfeder et al., 1995
; Aphasizheva et al., 1998
; Nixon et al., 1998
), DNA homology-independent recombination (Ostermeier et al., 1999
) and scaffold design based on combinatorial methods (Altamirano et al., 2000
) have been carried out for the generation of new protein lineages.
Here we present a method, designated functional salvage screen (FSS), to generate protein lineages with new sequence spaces through functional or structural salvage of a defective protein by employing green fluorescence protein (GFP) as a model protein. The functional salvage process started with a construction of the defective GFP expressing no fluorescence by genetically disrupting a predetermined region(s) of the protein. The defective template was designed to be unable to recover the functional trait (i.e. fluorescence emission) in vivo through simple insertion of base(s). Thus, only a recombination between a defective template in a predetermined region(s) and DNA segments derived from Escherichia coli chromosome could rescue the protein function. For the generation of a library of GFP variants from the defective template, two independent approaches, sequence-directed and PCR-coupled recombination, were attempted. The functionally salvaged, fluorescence-emitting variants with considerable stability were selected and analyzed with respect to sequence space and functional properties.
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Materials and methods |
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E.coli JM109 was used for the cloning and expression of the GFP variants. The pGFPuv vector (CLONTECH) was used as a source for the wild-type GFPuv gene. Plasmid pTrc-99A, used for library construction, was obtained from Amersham Pharmacia Biotech. The pMAL-c2 vector (New England Biolabs) was used to express the GFP variants as a fusion protein with the E.coli maltose binding protein (MBP). E.coli cells were grown at 37°C in LuriaBertani (LB) broth supplemented with ampicillin (50 µg/ml) when needed.
Construction of the defective GFP templates
The defective GFP templates were constructed as depicted schematically in Fig. 1. In order to remove the residue encoding 176V with additional one or two bases from the wild-type GFPuv, PCR was carried out using the primers, F1(5'-GCGAATTCAGTAAAGGAGAAGAACTTTTCAT-CGGA-3') and F3(5'-GCGGATCCATCTTCAATGTTGTGGCG-3') flanked by EcoRI and BamHI sites, respectively. The amplified DNA fragment was cloned into the pTrc-99A vector, yielding pGFPN. The wild-type GFPuv gene was again amplified by PCR with the following two sets of primers: F2(-1)(5'-GAGGATCCAACTAGCAGACCATTATCAAC-AAA-3')/F4(5'-AGTAAGCTTATTTGTAGAGCTCATCCAT-GCCATG-3') and F2(-2)(5'-GAGGATCCACTAGCAGACC-ATTATCAACAAA-3')/F4(5'-AGTAAGCTTATTTGTAGA-GCTCATCCATGCCATG-3'). Underlined sequences indicate the BamHI and HindIII sites, respectively. Each of the amplified fragments was inserted into the BamHI and HindIII sites of the pGFPN, resulting in pGFP
176(+1) and pGFP
176(+2), respectively. Two more templates, GFP
1723/176(+1) and GFP
1723/176(+2), were constructed from GFP
176(+1) and GFP
176(+2), respectively, by additional deletion of two residues 172E and 173D using PCR, according to a similar procedure to that described above. The resulting two constructs were designated pGFP
1723/176(+1) and pGFP
1723/176(+2), respectively.
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All the constructed templates were tested to confirm whether they are able to express the chromoprotein under various induction conditions by fluorescence microscopy (Abedi et al., 1998; Matz et al., 1999
).
Library construction for the functional salvage screen
A library for the functional salvage screen was constructed by two procedures, a sequence-directed (Jappelli and Brenner, 1999) and a PCR-coupled method (Kikuchi et al., 1999
). The sequence-directed process was basically similar to the shotgun cloning procedure. Four constructs containing each of the defective templates, pGFP
176(+1), pGFP
176(+2), pGFP
1723/176(+1) and pGFP
1723/176(+2), were linearized by digestion with BamHI and then eluted from agarose gel (0.8%). For oligonucleotide pools to be incorporated into the defective template genes, chromosomal DNA isolated from the E.coli MG1655 was digested with Sau3AI and the fragments ranging from 25 to 500 bp were eluted by using a DNA clean-up purification system (Promega). The resulting fragments were ligated with each of the previously linearized templates and then transformed into E.coli JM109 by electroporation.
For the PCR-coupled process, four defective GFPuv templates were amplified from each of the four constructs, pGFP176(+1), pGFP
176(+2), pGFP
1723/176(+1) and pGFP
1723/176(+2), by PCR using two primers, F1 and F4. The amplified fragments were cleaved by BamHI and then further digested with DNase I. The DNA fragments ranging from 50 to 150 bp were excised and eluted from agarose gel (2.5%) and then reassembled with the Sau3AI-digested chromosomal DNA (25500 bp) by PCR (94°C, 1 min; 45 ± 0.2°C/cycle, 1 min; 72°C, 40 s; total 40 cycles). The defective template (1020 ng/µl) was mixed with chromosomal DNA in ratios of about 1:1, 1:0.5, 1:0.1 and 1:0.01. The reassembled DNA fragments were amplified by PCR (94°C, 1 min; 50.5°C, 1 min; 72°C, 40 s; total 25 cycles) with two primers, F1 and F4. The resulting DNA fragments (0.52 kb) were purified, digested and cloned into the plasmid pTrc-99A and the constructs were transformed into E.coli JM109 by electroporation. To expand the mutation space, the stringency at the reassembling PCR step was modulated by either increasing or decreasing the annealing temperature.
For construction of a library from the template GFP129138/176(+2) through the dual-point salvage process, we incorporated the DNA fragments simultaneously into the two points (129D138G and 176V(+2) regions) of the template by using the PCR-coupled procedure as described above.
Screening of the functionally salvaged variants
Transformants were grown on agar plates in the presence and/or absence of IPTG and positive clones emitting fluorescence were first screened by direct observation under UV excitation (365 nm) using a hand-type UV lamp (Vilber Lourmat). As a control, E.coli cells harboring each of the defective templates were grown under the same conditions.
With the primarily isolated clones, we further screened the salvaged GFPs for structural stability in vivo as described in our previous work (Doi and Yanagawa, 1999). E.coli cells harboring the salvaged GFPs were cultivated in LuriaBertani medium at 37°C and induced with 0.2 mM of IPTG when the OD600 nm reached about 0.5. After 2 h of cultivation, chloramphenicol (100 µg/ml) was added to the medium to block further protein synthesis and an aliquot of 0.5 ml was removed at the indicated times and analyzed by SDSPAGE. A protein band corresponding to each of the functionally salvaged GFPs was scanned with a gel scanner. The clones showing a distinct protein band were isolated and the incorporated DNA segments were identified by DNA sequencing.
Protein purification and characterization
For the rapid purification and clear comparison with wild-type GFPuv, the genes encoding the selected GFP variants were subcloned into the EcoRI and HindIII sites of pMAL-c2 vector, expressed as MBP fusion proteins and purified (Kapust and Waugh 1999) according to the general procedure of the manufacturer.
The cleavage of fusion proteins was carried out to test the appropriate folding and thus accessibility of a site-specific protease factor Xa. During the reaction for 40 h at 810°C, aliquots were removed and analyzed by SDSPAGE, along with measurements of fluorescence intensity. The cleaved GFP variants were further purified by reapplying them on to the amylose resin and concentrated by dialysis against 20 mM TrisHCl (pH 8.0) buffer containing 5% glycerol.
Spectral properties of the salvaged GFPs were investigated at 25°C using a spectrofluorimeter (Amico). Bandwidths and integration times were kept at 5 nm and 0.5 s, respectively. Maximum emission and excitation wavelengths were first determined by automatic dual scanning and then confirmed by manual scanning at a predetermined wavelength. All scans were conducted in duplicate for various protein concentrations (5500 µg/ml).
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Results |
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The FSS process consists of the following steps as depicted in Figure 1. (i) A predetermined region(s) of a target protein, such as residue(s), domain(s) or module(s), is genetically disrupted by deletion, insertion, inversion or duplication, which results in a functionally defective protein. (ii) A library is generated from the defective gene by incorporation of the diverse nucleotide pool, such as fragmented E.coli chromosomal DNA, into the defined region(s) of the defective gene through either sequence-directed insertion or PCR-coupled recombination. (iii) The variants that recover the functional or structural trait are screened by the functional or genetic screening system. In step (i), any region(s) of the parental protein can be selected randomly or rationally. Step (ii) involves the incorporation of the segments, which can be either synthetic oligonucleotides or digested genomic DNA. The incorporation event is carried out through a sequence specific insertion resembling a shotgun cloning strategy (Jappelli and Brenner, 1999
) or homologous recombination using a PCR-like process (Kikuchi et al., 1999
). The critical point in the present strategy lies in designing a defective gene whose functional trait should not be recovered by simple insertion of base(s), which guarantees the generation of protein lineages with new sequence spaces.
Construction of the defective GFP templates for functional salvage screen
We employed a well-characterized green fluorescent protein (GFPuv) with enhanced fluorescence intensity as a model protein (Kim et al., 2000), which allows the rapid and simple detection of the functionally salvaged proteins from the defective one. A schematic procedure for construction of the defective GFP templates is described in Figure 1
. Two GFP variants, expressing no fluorescence, were first constructed by introducing functional defect into the parent GFPuv. Considering the fact that the deletion in the ß-strand of GFP (amino acid residues 175188) results in the loss of fluorescence (Li et al., 1997
), we carried out internal deletion in this region. The nucleotides of GFPuv gene (gttc and gttca) that encode 176V with successive one and two bases were deleted from the parent gene and the resulting templates were designated GFP
176(+1) and GFP
176(+2). In an effort to exclude the generation of variants salvaged by simple insertion of one or two base(s), we attempted further deletion of amino acid residues around the defective region and found that deletion of 172E and 173D leads to complete loss of fluorescence emission. Based on this finding, two more templates, GFP
1723/176(+1) and GFP
1723/176(+2), were constructed from GFP
176(+1) and GFP
176(+2), respectively, by additional deletion of the two residues 172E and 173D. This deletion is expected not only to expand the sequence space of the functionally salvaged GFPs, but also to eliminate the possibility that the defective GFPs might be salvaged by simple insertion of one or two base(s) countervailing the frame-shift mutations of GFP
176(+1) and GFP
176(+2).
All the constructed templates were tested as to whether they are able to emit the fluorescence. As a result, no fluorescence emission with excitation at 365405 nm was observed. As the deletions in the templates gave rise to the frame-shift mutations, the constructs were defective in both structural and functional aspects. The resulting templates, therefore, were considered to be suitable as the starting target genes for the functional salvage process.
Library construction and screening of the salvaged GFPs
In an effort to explore the feasibility of the functional salvage process and to obtain the diverse pool of salvaged GFPs, we constructed the libraries by applying two procedures, sequence-directed and PCR-coupled recombination, to each of the four defective templates, GFP176(+1), GFP
176(+2), GFP
1723/176(+1) and GFP
1723/176(+2). In the sequence-directed method, the oligonucleotide pool of Sau3AI-digested genomic DNA was incorporated into the target site. For construction of a more diverse library, we attempted a PCR strategy where a defective template, which had been cleaved with BamHI, was further digested with DNase I and reassembled with Sau3A1-digested chromosomal DNA. For both cases, the pool of the randomly digested genomic DNA ranging from 25 to 500 bp was used for the functional salvage process.
Total colonies ranging in number from 3500 to 6000 were obtained from each library and about 50 colonies were randomly picked and analyzed in terms of the percentage of oligonucleotide-inserted genes and size variation to investigate the genetic diversity of each library. As a result, about 70% of the colonies from the library generated by the sequence-directed method were found to possess the insert, whereas the library obtained by the PCR-coupled process revealed an insertion frequency ranging from 12 to 41%, depending on the template and stringency of PCR conditions used (Abecassis et al., 2000). With randomly selected colonies from the library constructed from GFP
176(+2) by the sequence-directed method, we analyzed the size variation of the genes. As shown in Figure 2A
, the size of the variants was found to be considerably diverse. The pool of genes generated from GFP
1723/176(+2) by the PCR-coupled method also revealed a random set of genes, as can be seen in agarose gel electrophoresis (Figure 2B
). When the library was constructed from GFP
1723/176(+2) under less stringent PCR conditions (lower temperature and rate of annealing), a pool with more diverse size variation was obtained, as shown in Figure 2C
. Similar trends were observed for the libraries constructed from the other templates (data not shown).
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Sequence diversity of the salvaged variants
From the library of GFP176(+2), 11 clones exhibiting different fluorescence intensities were randomly selected and analyzed. From them, we finally isolated nine clones having a distinct diversity based on restriction site analysis and SDSPAGE. With the identical procedure, we also chose 12 clones from the library of GFP
1723/176(+2). The genes from the selected clones were retransformed into a freshly prepared host (E.coli JM109) and grown in LB medium to confirm the formation of chromoprotein.
The amino acid sequences of the incorporated segments were deduced from DNA sequencing (Figure 3). Of nine clones from the library of GFP
176(+2), two variants had the same fragment with the GFPS7. In addition, two variants from the GFP
1723/176(+2) library were found to contain the identical sequence with the GFPI9 and GFP
13. It is likely that the sequence of the segments rescuing the defective GFPs was not biased, but randomly distributed. The site where the random fragment of genomic DNA is incorporated for functional salvage of the defective GFP was tolerable against the insertion of various segments, ranging from nine (GFPS7) to 52 amino acid residues (GFPI3). Apparently, comparison of the library from GFP
176(+2) with that from GFP
1723/176(+2) revealed that the sequence-directed salvage process results in more diverse lineages than the PCR-coupled method. It is worth noting that the incorporated segments of two libraries have different sequences, which strongly implies that further deletion in the template GFP
1723/176(+2) might affect the sequence space of the segment to be incorporated.
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Characterization of the functionally salvaged GFPs
For a rapid and clear comparison, the seven genes from the GFP176(+2) library were subcloned into pMAL-c2 vector to express them as MBP fusion proteins. Most of clones were also found to be fluorescent in the fusion state, except for the GFPS34, and similar results were obtained when induced at 37°C. From the SDSPAGE analysis, all of the MBP-fused proteins were mainly expressed in the soluble fraction (>60%) with calculated molecular masses and accounted for about 1015% of the total cell protein.
To investigate the functional properties of the salvaged GFPs, the fusion proteins were further purified to apparent homogeneity by using amylose resin. The two variants (Cont-1 and Cont-2) which emitted fluorescence but were excluded in the screening step because of their low structural stability were also expressed as fusion proteins and purified according to the same procedure for comparison. The purified fusion proteins showed different migration rates in SDSPAGE, depending on the size of the incorporated segments (Figure 4A). To get some insight into the functional expression of the fusion proteins, the purified proteins were treated with factor Xa and analyzed by SDSPAGE. As shown in Figure 4B
, seven fusion proteins of the salvaged GFPs were efficiently cleaved and separated into their respective domains, which indicates that both domains fold independently and are accessible to site-specific protease factor Xa. In the case of the two variants (Cont-1 and Cont-2), however, the respective domains were not detected when cleaved with factor Xa, as shown in lanes 3 and 4 of Figure 4B
. It is generally known that a misfolded or partly unfolded protein in the fusion state is structurally unstable and found to be tightly associated with bacterial chaperonin or protease. The result described here was the case that the two salvaged variants were partly misfolded and thus susceptible to protease, as reported by Keresztessy et al. (Keresztessy et al., 1996
). A similar result was observed in the control experiment using a general protease (Protease K). It is interesting that most of selected variants, when expressed in a single protein without MBP, emitted the fluorescence more persistently and retained their integrity than in the fusion state, and this might be attributed to a change in the fusion ability to proper folding in the salvaged variants with fusion partner MBP.
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For the functional characterization of the salvaged GFPs, the fluorescence properties of the selected variants were investigated (Table I). The wild-type GFPuv protein was first analyzed and excitation and emission maxima were observed at 397 and 508 nm, respectively. The functionally salvaged GFPs exhibited similar fluorescence properties, showing an excitation maximum at 395399 nm and an emission maximum at 506510 nm. The spectral properties were similar to each other, but the selected variants revealed a broad distribution of mean fluorescence intensity.
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In the natural evolutionary process of proteins, mutations occur randomly over the entire gene and the functional salvage process at multiple sites is expected to create more diverse protein lineages. In the multi-point salvage, the variants are supposed to recover their defective trait by complement at multiple sites. As a preliminary experiment to test the multi-point salvage, functional salvage at the dual-point was conducted. We designed a defective template in which an additional region (129D138G) of the GFP176(+2) was deleted and this template was designated GFP
129138/176(+2). The deleted region (129D138G) is part of a super-loop consisting of the residues from 129D to 142E (Ormo et al., 1996
; Yang et al., 1996.
) and also functionally indispensable (Li et al., 1997
). For the multiple site salvage, the PCR-coupled process might be more effective in generating the functionally salvaged GFPs, because it enables the incorporation of segments into multiple sites simultaneously. Thus, we obtained a library from GFP
129138/176(+2) by using the PCR-coupled process and picked clones randomly for analysis of their genetic diversity. As shown in Figure 5A
, more diversity in genotype of the GFP variants was observed compared with that produced by a single point salvage. To confirm the diversity at the protein level, the genes were expressed in E.coli and expressed proteins were analyzed by SDSPAGE (Figure 5B
). The GFP variants bearing the incorporated segments were predominantly detected and their size was found to be diverse, as anticipated from the genetic diversity.
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Discussion |
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It has been shown that GFPuv, with a nearly perfect shell around a chromophore by an 11-stranded ß-barrel, is generated through a complex process, leading to a highly compact and rigid structure for fluorescence emission (Ormo et al., 1996; Yang et al., 1996
). Thus, it has been considered that GFP is marginally tolerable against artificial transposition or insertion, displaying a vulnerable nature to structural change (Li et al., 1997
). It was also reported that deletions in either the internal or terminal region of GFP result in perturbation of the structural integrity and loss of fluorescence (Li et al., 1997
). Unexpectedly, nonetheless, a large number of functionally salvaged variants were generated by the functional salvage process, which indicates that this process provides an efficient route to create a pool of new protein lineages with different sequence spaces. In line with this, generation of the functionally salvaged GFPs supports the contention that the structural integrity of GFP can be further extended to the sequence space where further diversification is acceptable. Consistent with this view, experimental results have recently shown the expansion of sequence spaces in terms of structural and functional trait either in recombinant (Abedi et al., 1998
; Baird et al., 1999
) or natural GFP homologues (Matz et al., 1999
; Gross et al., 2000
; Lukyanov et al., 2000
; Wall et al., 2000
; Wiedenmann et al., 2000
).
The functionally salvaged GFP variants exhibited similar fluorescence properties in terms of maximum excitation and emission wavelengths when compared with the wild-type GFPuv, but their fluorescence intensities varied considerably. This result implies that the salvaged GFPs have different conformations or stabilities from the wild-type GFPuv, probably owing to the structural perturbation or subtle disorganization of the innate fluorophore by incorporation of segments. From a more detailed comparison, there appeared to be an apparent correlation between the portion of hydrophobic residues of the incorporated segments and fluorescence intensity. As observed in Figure 3 and Table I
, the GFPs salvaged with more hydrophobic residues produced less fluorescence than those harboring a short peptide or hydrophilic residues, resulting in an ~25100-fold lower intensity compared with the parent GFPuv. The salvaged variants, GFPS25, GFPS31, GFPI2 and GFP
10, contained a relatively high portion of hydrophobic amino acid residues in the segments compared with other variants and their fluorescence intensity was much lower than those of other variants. On the other hand, no apparent correlation was observed between the size of the incorporated segments and mean fluorescence value. The exact reason for these results remains to be elucidated, but it is plausible to suggest that the salvage point of the defective templates is tolerable to incorporation of diverse segments in a sequence-specific rather than a size-specific manner. Resolution of the structure of the functionally salvaged GFPs would demonstrate the effect of the sequence space of the rescuing segments on the properties of the resulting variants. Prior to structural resolution, it is also feasible that the salvaged variants might be further processed to improve their properties including stability and fluorescence intensity through in vitro evolution using random mutagenesis or DNA shuffling (Zhang et al., 1997
).
Considering the principle of the functional salvage process, four types of lineages are expected to be produced when the constructed templates were used, as depicted schematically in Figure 6. From the libraries generated from the four defective templates, 375 clones recovering the fluorescence through the salvage process were screened. Of these, 17 clones were randomly picked and typically characterized. As a preliminary result, random insertion of the segments was found to be a predominant event in the salvaged GFPs, mainly producing the type II lineage. In this work, screening of the salvaged GFPs relied on whether the green fluorescence is emitted at a single wavelength excitation (365 nm) and this might partly contribute to a narrow range of the salvaged variants (Matz et al., 1999
; Lukyanov et al., 2000
; Wiedenmann et al., 2000
). In fact, we also detected a significant number of type III and IV lineages based on the fluorescence emission and protein expression. Of the primarily screened clones, about 30% were analyzed to be salvaged by fusion at the external region (type III) and by early termination (type IV). Type III variants are likely to be mainly generated through a fusion of a large fragment, resulting in a fusion protein-like feature by structural rearrangement. On the other hand, type IV might be created by either an early termination due to the appearance of a stop codon or proteolysis in vivo. Production of type I lineage is expected when the defective template is constructed by deletion of either multiple sites or a larger fragment or domain, but this type could not be produced in the single point salvage process attempted mainly in this work.
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The functional salvage process relies on the screening of the protein variants with appropriately reorganized structures complementing a defective trait. Either non-essential or essential regions of a protein should be the target sites for the functional salvage screen and these sites can be deduced from various methods such as multiple alignments of the related proteins or comparison of their 3-D structures, which would lead to the development of a more rational strategy for the functional salvage screen. In this context, we believe that the functional salvage screen can be a valuable tool for the generation of protein lineages with new sequence spaces and the resulting variants can be further subjected to directed evolution to acquire the desired properties. The present approach may also be useful in studying the involvement of a specific region(s) or domain(s) in the structure and function of proteins.
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Notes |
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Received February 14, 2001; revised June 8, 2001; accepted June 18, 2001.