Department of Chemistry and Center for Fundamental and Applied Molecular Evolution, Emory University, 1515 Dickey Drive, Atlanta, GA 30322, USA
2 To whom correspondence should be addressed. E-mail: sal2{at}emory.edu
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Abstract |
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Keywords: directed evolution/intein/methodology/protein engineering/selection
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Introduction |
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In recent years, a variety of methods for identifying and removing frame-shifted DNA sequences have been developed. Initial approaches involved expressing the protein of interest as an N-terminal fusion to green fluorescent protein (Waldo et al., 1999) or chloramphenicol acetyltransferase (Maxwell et al., 1999
) (Figure 1A) to provide an observable phenotype (i.e. fluorescence or antibiotic resistance). While these methods were developed as intracellular assays for folding, primarily in response to the requirement for improved protein yields and solubility for structural genomics initiatives, the assumption a priori is that the reading frame must also be conserved. However, these systems are prone to identifying false positives due to initiation of translation at internal ribosomal binding sites and this lessens their utility in the context of directed evolution (Sieber et al., 2001
; Lutz et al., 2002
).
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While the dual reporter systems have proven efficient at removing frame-shifted library members, they also inevitably impart selection pressure for the folding and solubility of the protein of interest (Sieber et al., 2001; Lutz et al., 2002
; Zacchi et al., 2003
; Bittker et al., 2004
). For example, in pSALect an in-frame but aggregation-prone fusion protein would be unable to confer antibiotic resistance, especially as it has recently been shown that the Tat signal sequence does not export misfolded proteins (DeLisa et al., 2003
). Selection against misfolded or insoluble variants may be helpful in many directed evolution applications, as these clones are unlikely to be those that display the evolved property of interest. However, it is noteworthy that many functional variants identified by directed evolution suffer from an impaired ability to fold (Lee et al., 2003
). As it is impossible to quantify the degree of selection pressure for folding imparted by the existing systems, the risk is that functional variants will be eliminated in the course of pre-selection for reading frame. This is an especially pertinent concern for the SCRATCHY methodology (Lutz et al., 2001b
), in which reading frame selection is carried out after generating single-crossover hybrids, but before these variants are recombined to yield multiple-crossover clones (Figure 2). Clearly, single-crossover hybrids that misfold may still be able to contribute useful diversity to the final, multiple-crossover library; it would therefore be undesirable to eliminate them at the pre-selection stage.
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We have evaluated pInSALect by expressing in-frame and frame-shifted genes and by directly detecting the products of the splicing reaction. Additionally, libraries of hybrid genes created by incremental truncation (Lutz et al., 2001a) were subjected to reading frame selection in both pSALect and pInSALect and were subsequently assessed for biases in the crossover distributions of the selected variants that would reflect an underlying selection pressure for folding. The results demonstrated that pInSALect maintains the same, stringent selection for reading frame as pSALect, while eliminating undesired selection pressure for the folding of hybrid proteins.
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Materials and methods |
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Enzymes were purchased from New England Biolabs (Beverly, MA) unless otherwise indicated. Taq DNA polymerase was obtained from Promega (Madison, WI). DNA samples were purified using the QIAquick and QIAprep purification kits (Qiagen, Valencia, CA) according to the manufacturer's protocols. The protease inhibitor cocktail for use in the purification of histidine-tagged proteins and all antibodies for immunoblotting were purchased from Sigma (St Louis, MO). Primers were purchased from Integrated DNA Technologies (Coralville, IA). All plasmid modifications were confirmed by DNA sequencing.
Construction of pInSALect
The gene encoding the VMA intein (GenBank No. AB093499) was amplified by polymerase chain reaction (PCR) from S.cerevisiae (commercial sample of baker's yeast) using oligonucleotides ScIN.for (5'-GCG ATT AAT GGG TGC TTT GCC AAG GGT ACC-3') and ScIC.rev (5'-CGC GCT AGC GCA ATT ATG GAC GAC AAC CTG-3'), which also introduced restriction sites for AseI and NheI (underlined). The resulting 1386 bp fragment was digested with AseI and NheI and cloned into the NdeI/SpeI sites of pSALect (Lutz et al., 2002), which have compatible cohesive ends but result in a ligation product that cannot be recleaved. Next, overlap extension PCR (Horton et al., 1990
) was used to introduce unique restriction sites for NdeI and SpeI into the endonuclease domain of the intein, between amino acid resides Gly273 and Gly275 (numbered according to amino acid position in the S.cerevisiae intein, beginning with Cys1 and ending with Asn454). Primary amplifications were performed with the following primer pairs: ScIN.for with 5'-ACC ACT AGT ACC ACC CAT ATG ACC TCT GAC AAC TTT AGA GTA-3' (5' fragment) and 5'-CAT ATG GGT GGT ACT AGT GGT ATT CGC AAT AAT CTT AAT ACT GAG-3' with ScIC.rev (3' fragment). The primer-encoded restriction sites are underlined. The full-length intein was reassembled by PCR with primers ScIN.for and ScIC.rev and the product was re-inserted into pSALect after digestion with AseI and NheI. This strategy resulted in the replacement of Asn274 of the parental intein sequence with the linker HisMetGlyGlyThrSer.
Construction of test plasmids
The D.melanogaster deoxyribonucleoside kinase (DmdNK) gene was amplified by reverse transcription of embryonic mRNA (kindly provided by Professor Susan Abmayr, Pennsylvania State University, University Park, PA) using Qiagen's OneStep RT-PCR kit with primers 5'-CGC CAT ATG AAG TAC GCC GAG GGC ACC CAG-3' and 5'-GCG GAA TTC CTC GAG ACT AGT TCA GGG CTG TTG GTT ACT TGA-3'. The resulting PCR product was digested with NdeI and XhoI and ligated with pET-16b (Novagen, Madison, WI). The reverse primer also introduced an SpeI restriction site (underlined) into the pET-16b vector.
The gene encoding human mitochondrial thymidine kinase 2 (hTK2) was isolated from a human pancreas cDNA library (Marathon-Ready, Clontech, Palo Alto, CA) by PCR with primers 5'-CGC CAT ATG TCA GTG ATC TGT GT CGA GGG C-3' and 5'-CGC ACT AGT TCA TGG GCA ATG CTT CCG ATT CTC TGG-3'. The underlined NdeI and SpeI restriction sites were used to sub-clone hTK2 into pET-16b. The hTK2 gene was corrected for codon usage in Escherichia coli; the rare arginine codons AGA and AGG were mutated to CGC at amino acid positions R79, R93, R99, R107, R129, R151, R155 and R157.
For sub-cloning into pSALect and pInSALect, the stop codon of each gene was replaced by a GGA triplet (underlined below), creating a glycine linker between the test protein and ß-lactamase, using the forward primer T7 (5'-TAA TAC GAC TCA CTA TAG GG-3') and the reverse primers 5'-CGC ACT AGT TCC GGG CTG TTG GTT ACT TGA GAT-3' (DmdNK) or 5'-CGC ACT AGT TCC TGG GCA ATG CTT CCG ATT CTC-3' (hTK2). Frame-shifted variants of DmdNK and hTK2 were constructed by a single nucleotide insertion (underlined) in the reverse primers (DmdNK, 5'-CGC ACT AGT TCC GTG GCT GTT GGT TAC TTG AGA T-3'; hTK2, 5'-CGC ACT AGT TCC TCG GGC AAT GCT TCC GAT TCT C-3'). The PCR products were digested with NdeI and SpeI and ligated into pSALect and pInSALect.
Construction of pBlaFla
For western immunoblotting analyses, the FLAG tag sequence (DYKDDDDK) was introduced at the C-terminus of the pInSALect-encoded fusion protein by PCR amplification with primers 5'-CAT ATG GGT GGT ACT AGT GGT ATT CGC AAT AAT CTT AAT ACT GAG-3' and 5'-CGC GAA TTC TCA CTT GTC GTC ATC GTC CTT GTA GTC TCC CCA ATG CTT AAT CAG TGA GGC-3' (FLAG tag sequence underlined). The 1395 bp product was digested with SpeI and EcoRI and cloned into the corresponding SpeI/EcoRI sites of pInSALect.
Restreaking test
Escherichia coli DH5-E (Invitrogen, Carlsbad, CA) were transformed with each plasmid, plated on LBagar plates containing chloramphenicol (Cm; 50 µg/ml) and incubated at 37°C. Single colonies from each transformation were used to inoculate 2 ml cultures of LBCm (50 µg/ml). After overnight growth at 30°C, LBCm medium was used to dilute each culture to A600 = 0.2. An aliquot (4 µl) of each normalized culture was plated on LBagar containing carbenicillin (Carb; 100 µg/ml) and, as a control, on Cm-containing plates. Plates were incubated at 22, 30 and 37°C.
Solubility test
Escherichia coli BL21(DE3)pLysS cells (Novagen) transformed with pET-16b(DmdNK) and pET-16b(hTK2) were grown at 37°C in 50 ml LB broth containing Carb (100 µg/ml) and Cm (50 µg/ml) to an A600 of 0.6. Induction was for 3 h at 30°C in the presence of 1 mM isopropyl-ß-D-thiogalactopyranoside. The cells were harvested by centrifugation and the resulting cell pellets were each resuspended in 5 ml of lysis buffer (50 mM potassium phosphate pH 8, 300 mM NaCl, 10 mM imidazole, 25 µl protease inhibitor cocktail). An aliquot of the resuspended cells was collected for SDSPAGE analysis of the total cell fraction. Lysozyme was added to a final concentration of 0.1 mg/ml and the bacteria were lysed by sonication on ice. Benzonase nuclease (Novagen) was added to reduce the viscosity of the lysate and the sample was centrifuged for 30 min at 4500 g. The clarified supernatant was collected for the analysis of soluble cellular protein. The remaining pellet was resuspended in 5 ml of lysis buffer for analysis of the insoluble fraction. Samples were analyzed by SDSPAGE.
In vivo protein splicing assays
Transformed E.coli DH5-E were grown to mid-log phase in LB medium containing Carb (100 µg/ml) and harvested by centrifugation. The soluble periplasmic fraction was obtained by the method of Neu and Heppel (1965)
except that an ice-cold solution of MgSO4 (5 mM) was used in place of pure water to resuspend the cell pellet. After collecting the cold osmotic shock fluid, it was buffered by the addition of TrisHCl (pH 7.5; final concentration 20 mM) and concentrated using Amicon Ultra-4 spin filters (10 kDa molecular weight cut-off; Millipore, Billerica, MA). Immunoblots utilized the anti-FLAG M2 monoclonal antibody and were developed colorimetrically using a peroxidase-conjugated anti-mouse IgG with the SigmaFast peroxidase substrate tablet set.
Library construction
This study employed the library of purN and hGART single-crossover hybrids (the PGX library) previously constructed for validating the pSALect system (Lutz et al., 2002). Hybrid genes were excised from pSALect by digestion with NdeI and SpeI and ligated directly into pInSALect that had been treated with the same enzymes. The products were desalted by ethanol precipitation and used to transform E.coli DH5
-E by electroporation. Cells were plated on LBagar medium containing Cm (50 µg/ml) and, after 12 h at 37°C, colonies were recovered in 2 x YT medium supplemented with glucose (2%, w/v) and glycerol (15%, v/v). Reading frame selection was carried out by replating the pSALect and pInSALect libraries on LBampicillin (100 µg/ml) and incubating at 21°C. Colonies appeared after 7296 h and were analyzed by DNA sequencing.
The inverse incremental truncation library, consisting of hGARTpurN (GPX) hybrid genes, was also investigated in the two reading frame selection systems. This library had been characterized previously (Lutz et al., 2001b). The GPX library was removed from pDIM (Lutz et al., 2001a
) by digestion with NotI and SpeI and the reaction products were separated on a 2.5% (w/v) agarose gel. Hybrid genes of approximately parental size were excised from the gel and recovered using QIAquick spin columns. Library amplification was by PCR using the forward primer 5'-ATA GAT TTC AAG GAG ACA GTC CAT ATG-3' and the reverse primer 5'-GCA CTA GTT CCC TCG TCG GCA GC-3', with the underlined sequence replacing the stop codon from pDIM-GPX with a codon for glycine. The PCR products were restricted with NdeI and SpeI and ligated into the corresponding sites in pSALect and pInSALect as described above. Reading frame selection for both GPX libraries was performed as described for the PGX libraries.
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Results and discussion |
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To construct a reading frame selection system without protein folding requirements, we redesigned pSALect through the introduction of the cis-splicing VMA intein, producing pInSALect. The VMA sequence was isolated with gene-specific primers from commercially available baker's yeast. The full-length intein includes a homing endonuclease domain (Chong et al., 1996), which we initially removed in order to minimize the size of the fusion protein encoded by pInSALect. While this domain is not required for protein splicing (Chong and Xu, 1997
), its absence led to significantly impaired cell growth under selection conditions. We therefore incorporated the entire intein sequence in the pInSALect vector. The restriction sites for cloning genes of interest were engineered into the same loop of the endonuclease domain that had previously been shown to tolerate insertions without affecting splicing (Chong et al., 1998a
,b
).
An important element in the design of pInSALect was the incorporation of the Tat signal sequence. While the use of this signal sequence has been discussed in the context of pSALect (Lutz et al., 2002), it offers additional advantages for pInSALect. In requiring the completion of translation and protein folding prior to export (Yahr and Wickner, 2001
; DeLisa et al., 2003
; Palmer and Berks, 2003
), the Tat pathway allows splicing to take place in the cytoplasm. In contrast, translocation by the Sec pathway can be either co- or post-translational, depending on the nature of the protein (Lee and Beckwith, 1986
). Unbiased reading frame selection using the latter pathway could then rely on splicing in the comparatively oxidizing environment of the periplasm, the feasibility of which remains untested. Furthermore, the comparatively poor efficiency of protein translocation by the Tat pathway (DeLisa et al., 2004
) is advantageous, as it ensures that fusion proteins remain in the cytoplasm long enough for the splicing reaction to occur.
Vector validation
The primary consideration with our new system was to ensure that it imparted a reliable selection for maintenance of reading frame. To test this, the D.melanogaster deoxynucleoside kinase (DmdNK) gene (Johansson et al., 1999) was inserted into the NdeI/SpeI cloning site of pInSALect, generating pInSALect(DmdNK). A frame-shifted version with a single nucleotide insertion in the last codon of the gene was constructed as a negative control. When E.coli harboring pInSALect(DmdNK) were plated on LBcarbenicillin medium, colony growth was observed within 24 h at temperatures of 22 or 30°C. In contrast, no growth was observed under any conditions (22, 30 or 37°C; incubation times of up to 10 days) on plates with the frame-shifted gene in pInSALect. The results indicated that, as expected, disruption of the reading frame in a gene of interest is not compatible with survival of the host cell. These data were consistent with those previously reported for pSALect (Lutz et al., 2002
). More importantly, the experiment demonstrated that the bacterial host could express a functional version of the extended fusion protein (763 amino acids plus the target protein), provided the correct reading frame was maintained.
The human mitochondrial thymidine kinase (hTK2) gene was cloned into pInSALect to assess whether it truly provided reading frame selection without a requirement for protein folding. This protein had previously been reported to form inclusion bodies when it was over-expressed in E.coli, unless the GroEL/ES system was co-expressed and a heat-shock response was induced (Barroso et al., 2003). Our own over-expression experiments confirmed that hTK2 partitioned into the insoluble fraction, in contrast to DmdNK, which was expressed solubly (Figure 3A). We hypothesized that intein-catalyzed removal of insoluble hTK2 from the pInSALect-encoded fusion protein would enable growth under selection conditions, while the fusion protein expressed from pSALect(hTK2) would not confer antibiotic resistance. Plating the two clones on LBcarbenicillin confirmed that this was the case (Figure 3B). A variant that incorporated a +1 frame-shift in hTK2 was not viable under selection conditions in either pSALect or pInSALect (Figure 3B). Critically, then, it was shown that misfolding proteins such as hTK2 could be positively selected by the pInSALect system provided they maintained a correct reading frame, while pSALect would eliminate such clones owing to their insolubility.
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Libraries of single-crossover hybrids of the glycinamide ribonucleotide formyltransferases from E.coli (purN) and human (hGART) (Lutz et al., 2001a,b
, 2002
) were used to evaluate the ability of pInSALect to identify in-frame clones in the context of a directed evolution experiment. The purN-hGART (PGX) and hGART-purN (GPX) libraries were subcloned into both pInSALect and pSALect. Based on colony counts when grown on LBchloramphenicol medium, the sizes of the four libraries were estimated to be 9.7 x 104 clones for pInSALect-PGX, 1.0 x 106 for pInSALect-GPX, 6.4 x 104 for pSALect-PGX (Lutz et al., 2002
) and 9.1 x 104 for pSALect-GPX.
After reading frame selection on LBampicillin medium, sequences from a total of 167 colonies (GPX, n = 78; PGX, n = 89) were analyzed. Only one sequence (in the pSALect-PGX sample) was out of frame at the crossover point. We hypothesized that this false positive was a result of ampicillin hydrolysis over the extended selection period (up to 96 h); the use of carbenicillin is therefore preferable. These results demonstated that each vector efficiently removed out-of-frame sequences from the libraries; however, they offered no insights into whether in-frame sequences were also being excluded.
Unbiased reading frame selection
With pInSALect, we aimed to design a system that was strictly selective for reading frame, but that was not influenced by whether the protein of interest could fold. In such a system, one would expect the percentage of clones surviving reading frame selection to equal the percentage of the naïve (i.e. pre-selection) library that was in frame. Sequence analysis of colonies from the naïve libraries (GPX, n = 63; PGX, n = 66) showed that 14% (GPX) and 38% (PGX) respectively were in frame (Table I). These percentages serve as baseline values for comparison with survival rates on reading frame selection. In the case of pSALect, plating the libraries under selection conditions led to a dramatically reduced survival rate (Table I). These data suggested that only one in five in-frame hybrids survived reading frame selection from the pSALect-PGX library; the figure was closer to one in 13 for the pSALect-GPX library. The implication is that the pSALect system applies strong selection pressure beyond a requirement for maintenance of reading frame, resulting in the removal of up to 90% of in-frame hybrids because of their misfolding or limited solubility. In contrast, the proportions of cells surviving selection in the pInSALect libraries closely matched the percentages of in-frame clones in the naïve libraries. Selection in pInSALect therefore appears to be limited to reading frame alone.
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The crossover positions in each library are clearly not normally distributed. We therefore employed the KolmogorovSmirnov (KS) test to compare the distributions of crossovers in naïve, pSALect and pInSALect libraries. The KS test compares cumulative percentile plots (Figure 6) and generates a test statistic, D, corresponding to the maximum difference between two cumulative distributions. This in turn allows calculation of P, the significance of the difference (Press et al., 2002). The D and P values, when each of the GPX and PGX libraries were compared, are summarized in Table III. The statistics confirmed that using pSALect for reading frame selection imparts a significant bias on the distribution of crossovers in the resulting library. This is most evident for the GPX library, where a P value of <5 x 104 corresponds to >99.95% confidence that the naïve and pSALect libraries are different. In contrast, both pInSALect libraries show crossover distributions that are statistically indistinguishable from the naïve datasets. Notably, crossovers in the naïve GPX library are biased towards the N-terminal half of the hybrid protein. The pInSALect system faithfully maintains this skew in the process of eliminating frame-shifted clones.
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The qualitative and quantitative data presented here confirm that the pInSALect system reliably selects genes with the desired reading frame, while exerting no additional selection pressure for the folding or solubility of the resulting protein. By encoding a self-excising intein that also removes the protein of interest, export of ß-lactamase to the periplasm becomes solely dependent on translation of an in-frame gene. Our experiments confirm that splicing does occur prior to export and that pInSALect is superior to pSALect for unbiased reading frame selection of combinatorial libraries.
We envision that pInSALect will find a number of applications in protein engineering and functional genomics. In the SCRATCHY methodology, the premature removal of a subset of in-frame hybrid genes is undesirable because it reduces diversity for the subsequent DNA shuffling step; pInSALect has therefore become an integral component of our protein engineering strategies. Although not intended for protein over-expression, additional applications of pInSALect could include the analysis of genomic or cDNA libraries, providing a convenient pre-screen for codon-optimized open reading frames. Moreover, pInSALect can be used to maintain folding-compromised protein variants in E.coli, effectively functioning as a pre-screen for reading frame alone. These variants may then be transferred to a non-bacterial host for protein expression or may become new targets for directed evolution approaches that improve heterologous expression and/or solubility. Finally, pInSALect offers a means to begin the directed evolution of proteins that have traditionally proven troublesome, such as membrane proteins and those that are insoluble in the absence of oligomerization partners.
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Notes |
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Acknowledgments |
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References |
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Received June 23, 2004; revised August 3, 2004; accepted August 13, 2004.
Edited by Andreas Plueckthun