Improving a circularly permuted TEM-1 ß-lactamase by directed evolution

Joel Osuna,1, Alejandra Pérez-Blancas and Xavier Soberón

Instituto de Biotecnología, UNAM, Apdo. Postal 510-3 Cuernavaca, Morelos 62250, México


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Circular permutation of proteins is a powerful technique to explore the importance of the polypeptide secondary structure order for attaining the final three-dimensional structure. Here, we designed a circular permutation of the TEM ß-lactamase in order to produce a new domain-forming amino acid arrangement in the polypeptide sequence. Closing the normal N- and C-termini with the connecting peptide GGS and creating new N- and C-termini at position 216, produces a severely impaired permuted protein. Introduction of a connector with random components allows the isolation of enzymes with better activities and indicates a selection for a potential helix-stop signal at the new super-secondary motif. We applied several directed-evolution cycles, starting from permuted enzymes with each of the two different connecting peptides, and selecting for antibiotic resistance and isolated several mutants with resistance levels close to those of the wild-type enzyme. We also analyze some of the data collected on the outcomes and paths of these evolutionary experiments. A purified sixth cycle variant with connector peptide GGS showed catalytic efficiency values ~8% of the natural enzyme.

Keywords: circular permutation/directed-evolution/TEM ß-lactamase


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Since the first reported circular permutation experiment in a protein by Goldenberg and Creighton (Goldenberg and Creighton, 1983Go), a number of circularly permuted proteins have been constructed by genetic engineering methods (Viguera et al., 1995Go; Luger et al., 1989Go).

In most cases, the native N- and C-termini have been joined by flexible polypeptide linkers and the new termini usually introduced in solvent exposed loops in order to retain protein structural and functional integrity.

The results from these studies demonstrate that the tendency to attain a stable conformation is not limited by the order of the secondary structural elements in the polypeptide sequences. Furthermore, recent random circular permutation experiments show that new termini are even tolerated within regular secondary structures (Graf and Schachman, 1996Go; Baird et al., 1999Go; Hennecke et al., 1999Go; Iwakura et al., 2000Go).

The class A ß-lactamase fold (Jelsch et al., 1993Go) is a good model protein where circular permutation of the polypeptide chain can be used to untangle two intimately intertwined domains (Figure 1Go). The class A ß-lactamase modular structure results from two crossovers of the polypeptide chain from one domain to the other (Figure 1Go). The final result produces a ß-sandwich-like domain that contains amino acid residues from both the N- and C-terminal parts of the protein and a mostly helical domain that contains amino acid residues from the central part of the polypeptide chain. In addition, TEM ß-lactamase has been used as a model protein in numerous works and in particular in Stemmer's classical directedevolution experiments (Stemmer, 1994Go) and more recently (Zaccolo and Gherardi, 1999Go) has been used for testing more aggressive directed-evolution approaches.



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Fig. 1. Three-dimensional structure of TEM ß-lactamase. The figure was made with the VMD program (Humphrey et al., 1996Go). The old N- and C-termini are indicated. The new termini were designed at the second protein crossover. The catalytic residue Ser70 is indicated to show the active site position in the protein.

 
In this work, we attempted the construction of a circular permutation of the TEM ß-lactamase with new termini located at the second crossover. This will produce a protein with a new linear arrangement of the domain-forming amino acid residues in the polypeptide sequence. Additionally, due to the close proximity of the second crossover to the catalytic site, an extra challenge was expected in order to obtain a functionally proficient permuted variant. We also take this model as a good test bed to explore what is likely to be a general phenomenon, namely the recovery of function from a strongly perturbed structure, as it comes out of an initial selection for a particular activity.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Materials

Oligonucleotides were synthesized in an in-house facility (Instituto de Biotecnología). Restriction endonucleases and T4 DNA ligase were purchased from New England Biolabs (Beverly, MA) and Taq Gold DNA polymerase from Perkin-Elmer (Branchburg, NJ). Nitrocefin was purchased from Becton Dickinson (Cockeysville, MD) and all other antibiotics from Sigma Chemicals (St. Louis, MO).

Strains, plasmids and general DNA procedures

Strain XL1-Blue (Stratagene) was used for routine cloning, DNA plasmid purification and protein expression. Plasmid and PCR product purification were made according to kit manufacturers (Boehringer, Mannheim, Germany). Other DNA manipulations were made according to standard procedures. DNA sequencing was performed in an in-house facility (Instituto de Biotecnología). Plasmid pT4, containing a constitutive Trc promoter and a kanamycin marker, was used for the cloning and expression experiments (kindly provided by Peter Kast and Ying Tang, ETH-Zurich, Switzerland). Plasmid pT4-Bla is identical to the previous one but the Trc promoter was replaced with the TEM ß-lactamase gene promoter.

ß-Lactamase-permuted gene construction

The ß-lactamase-permuted gene was constructed in three steps as follows (Figure 2Go). The first step used the oligonucleotides B45 (5'-TTTGCGGCATTTTGCCTTCCTGTTTTTGCTGGACCACTTCTGCGC-3') and A35 (5'-TTCACCAGCGTTTCTGGGTGGGATCCACCCTTAATCAGTGAGGC-3') to carry out PCR reaction A using pUC vectors (New England Biolabs) as a DNA template. The segment of B45 oligonucleotide that codes for the last 10 amino acids of the normal TEM ß-lactamase signal peptide is indicated in bold face and the rest of B45 is complementary to the gene region coding for the amino acid positions 218–222 of the mature protein. The different portions of the A35 oligonucleotide are as follows: the 5'-end (bold face) codes for the positions 26–32 of the mature protein; the other segment (underlined) codes for the linker peptide GGS (in the case of the random linker oligonucleotides, this part is replaced for a run of three, four or five NNG/C codons); and the italic part codes for the positions 284–288 of the mature protein. The second step used oligonucleotides D21 (5'-CACCCAGAAACGCTGGTGAAA-3') that is complementary to the gene region that codes for positions 26–32 of the mature protein, and C37 (5'-CCGGCTCGAGTCATTAGCCTTTGTTCGCCTCCA-TCCA-3') which is complementary to the gene region coding for positions 210–216 of the mature protein (3'-end, bold face) with alterations to the original nucleotide sequence (indicated by italics; D214->Asn and V216->Gly mutations are introduced by the oligonucleotide). Two stop codons are added after position 216. A XhoI restriction site (underlined) is added for cloning purposes. D21 and C37 oligonucleotides were used for PCR reaction B, with pUC vectors as DNA template. Both PCR products A and B were gel-purified and used in a third step PCR reaction (without pUC DNA template) and the expected full-length PCR product amplified with oligonucleotides Nco-SP (5'-GGGCCATGGCTATTCAACAT TTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTT-3', needed to complete the signal peptide and to add the restriction site NcoI for cloning purposes) and C37. To introduce the NcoI restriction site it was necessary to mutate (Ser->Ala) the first codon of the signal peptide (as indicated in italics). The final PCR product was gel-purified, digested with restriction enzymes NcoI and XhoI and cloned in the pT4 vector already digested with the same restriction enzymes.



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Fig. 2. Scheme of the construction of the circularly permuted gene of TEM ß-lactamase. The indicated oligonucleotides are described in Materials and methods. Vertical hatching indicates the signal peptide. The horizontal hatching indicates the region (position 26–31 of the mature protein) where PCR products A and B overlap. The diagonal hatching indicates the linker region. The final PCR reaction completes the signal peptide and introduces the NcoI and XhoI restriction sites needed for cloning.

 
In vitro evolution

In vitro evolution was done essentially as described by Stemmer (Stemmer, 1994Go). The circularly permuted genes were amplified with the oligonucleotides B45 and C37. The amplification products (~8 µg per reaction) were DNase-I digested (0.01 U) (Boehringer) for 2–10 min in five separate reactions at room temperature. The reactions were loaded onto a 2% agarose gel and fragments between 50 and 300 bp were gel-purified. The purified DNA fragments were used for gene reassembly without primers in different reactions containing from 500 to 2000 ng of DNA. One microliter (1/100) aliquots of the previous reactions were used as templates for a PCR reaction with primers Nco-SP and C37. The final PCR products were digested with NcoI and XhoI and cloned into pT4 vectors. XL1-Blue cells were transformed by electroporation with the respective ligation reaction, yielding approximately 1–3x106 unique clones in a single electroporation. Transformed cells were plated on LB kanamycin–ampicillin plates, and incubated at 30°C for 24 h.

Determination of ampicillin minimum inhibitory concentration (MIC) values

Five microliters of an overnight culture of XL1-Blue harboring the different evolved variants were spotted after a 10-3- or 10-5-fold dilution (10–50 colonies appeared without ampicillin in the last dilution) with fresh LB medium onto LB kanamycin plates containing increasing concentrations of ampicillin. The minimum concentration of ampicillin completely inhibiting the growth of cells was taken as the MIC.

Protein expression, isolation and purification

The GGS sixth cycle variant was expressed and purified following the procedure described by Lietz et al. (Lietz et al., 2000Go). Briefly, 1 l of LB medium was inoculated using a 10 ml starter culture. Cells were harvested by centrifuging at 8000 rpm and the pelleted cells were washed by resuspending in 100 ml of cold 10 mM Tris–HCl (pH 8.0)–30 mM NaCl buffer. The washed cells were centrifuged at 8000 rpm and the pellet resuspended in 50 ml of 20% sucrose, 30 mM Tris–HCl, pH 8.0, 1 mM EDTA. The cells were centrifuged at 8000 rpm and the pelleted cells were osmotically shocked by gently resuspending in 50 ml of cold deionized water. The pH of the osmotic shock supernatant was lowered to 4.8 by the addition of 25 ml of 0.002 M NaOAc (pH 4.8)–0.02 M NaCl. The precipitate was removed by centrifugation at 7000 r.p.m. for 20 min. The supernatant was loaded onto a previously equilibrated 1x5 cm CM-Sepharose column. The column was rinsed with 0.02 M NaOAc (pH 4.8)–0.02 M NaCl buffer until the absorbance at 280 nm returned to the base-line and then eluted with a salt gradient (0.02–0.5 M). Fractions containing ß-lactamase activity, as shown by a nitrocefin visual assay, were pooled and exchanged into 0.05 M phosphate buffer (pH 7.0) by dialysis. The protein concentration was determined by the method of Bradford (Bradford, 1976Go). The protein yield for the purified protein was ~2.5 mg/l of culture.

Enzyme kinetics and determination of specific activity of evolved variants

ß-Lactamase specific activity was determined by measuring the rate of nitrocefin hydrolysis at 482 nm at 25°C in 50 mM sodium phosphate buffer (pH 7.0) (O'Callaghan et al., 1972Go). Protein expression for all the variants, including the normal wild-type enzyme, was under control of the Trc promoter. Protein concentration in the cellular extracts was determined by the method of Bradford. Spectrophotometric measurements were made in a Beckman DU650 spectrophotometer using a 1.0 cm pathlength cuvette. Specific activity is reported as mM of product formed per mg of total protein per min. The sixth cycle GGS variant activity toward benzylpenicillin was followed at 240 nm ({varepsilon} = 820 M-1 cm-1; {Delta}{varepsilon} = 570 M-1 cm-1) by measuring the initial rates at different substrate concentrations. The kinetic parameters were determined by plotting initial rates versus [S], and the data fitted to the differential form of the Michaelis–Menten equation with Kaleidagraph (Synergy Software, Reading, PA, USA).


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Design of the circularly permuted TEM ß-lactamase

At the beginning of this work, our strategy focused mainly on the design of the new connection between the N- and C-termini. The TEM ß-lactamase fold contains an {alpha}-helix at the start and end of the molecule, perfectly fitting the described characteristics for an {alpha}{alpha} hairpin but lacking the inter-helical loop. We decided to make a shorter C-terminal {alpha}-helix [truncated at position Lys288, Ambler et al. numbering (Ambler et al., 1991Go)] to allow the connecting peptide for a better approach to the N-part (without modifications) of the partner helix as suggested from the detailed analysis of {alpha}{alpha} hairpins described by Efimov (Efimov, 1991Go). We reasoned that a short connection Gly–Gly–Ser could be enough for allowing a stable formation of the new super-secondary unit.

To create the new molecule termini at the second crossover region, we chose to cleave the protein at the inter-helical portion that separates {alpha}-helix {alpha}9 from {alpha}10. We chose position 216 as the new C-terminus and position 218 as the new N-terminus, and made some modifications at the cleaving site: residue Val216 was substituted with Gly, and residue Ala217 was deleted, in order to create some room needed to release possible steric interference with the rest of the molecule. We also modified residue Asp214 to Asn, since it has been proposed (Jelsch et al., 1993Go) that aspartate at position 214 might be found in a protonated state influenced by a nearby carboxylate residue (Asp233) in the wild-type structure. Other than the previous considerations, our choice of new N- and C-termini is somewhat arbitrary.

When produced in Escherichia coli, the designed circular permutation of TEM ß-lactamase was able to confer very low ampicillin resistance to bacteria (~5–6 µg/ml) even when the protein expression was controlled with a moderately strong promoter (Trc promoter). Furthermore, we noted some possible toxic effects of the designed construction as evidenced by a smaller size of the colonies producing the circularly permuted protein as compared with cells producing the wild-type enzyme plated on non-ampicillin culture media.

Pieper et al. (Pieper et al., 1997Go) described the construction of three circular permutations of the Staphylococcus aureus PC1 ß-lactamase. The most active permuted variant consisted of a construct cleaved in a loop far from the second crossover. However, two of the permuted proteins with new termini at the second crossover showed impairments in the activity and stability of the protein as compared with the wild-type enzyme. The permuted protein with a cleavage at the second crossover in a position similar to ours (the new termini were at position 211 instead of position 216 as ours and the connecting peptide was also different to our design) was reported as a severely impaired enzyme variant showing very low expression levels probably as a result of degradation problems. Our results, as well as those of Pieper et al. (Pieper et al., 1997Go) reveal the importance of modifications at the second crossover for the function and stability of the permuted protein.

Due to the above results, we decided to tackle the problem following two different strategies. First, searching for the optimal connecting peptide using selection for antibiotic resistance of bacteria transformed with a library of permuted genes containing variable peptide connectors of three, four or five amino acids long, and second, looking for improvement of the designed circularly permuted protein described above using directed-evolution strategies.

Random variability in the connecting peptide

We produced three different libraries with connecting peptides three, four or five amino acids long. The libraries were cloned in a vector containing the Trc promoter, as it is responsible for the expression of the different permuted genes, and selection in a range from 40 to 200 µg/ml ampicillin was used in order to isolate functional variants.

As shown in Table IGo, several potential functional candidates were found in all three libraries. However, the candidates able to confer higher resistance levels to bacteria were only found in the library containing the five amino acid connecting peptide.


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Table I. Linker amino acid sequences of functional variants andß-lactamase-specific activities
 
Analysis of Table IGo clearly shows a preference for a tryptophan residue at the second position of the connecting peptide. This result is easily explained in the light of the three-dimensional structure of TEM ß-lactamase (Jelsch et al., 1993Go). In TEM ß-lactamase, the tryptophan at position 290 is closely involved in several contacts with part of the ß-sheet. Removal of residue Trp290 could conceivably produce an unstable protein variant due to the loss of these contacts. It was interesting to find such relevance for position W290, since analysis of the Class A amino acid sequence alignment showed a preference for a hydrophobic residue at that position. This could point for a particular fine-tuning of the structural role played by W290 in the TEM protein as compared with other protein family members.

As shown in Table IGo, at position 1 in the variable connecting peptide we preferentially found a Gly or Asn residue. The residue preference at this position could be explained if we assign a Ccap role for these amino acids. It is known that both the Schellman and {alpha}L helical capping motifs show some preference for Gly, His or Asn at the Ccap position (Aurora et al., 1994Go; Kumar and Bansal, 1998Go).

Finally, we observe a rather large variability at positions 3, 4 and 5 of the variable connecting peptide, with some preference for charged residues. This could be useful to stabilize the partial charges developed at the start and end of {alpha}-helices.

In order to analyze the resistance level of bacteria containing one of the best clones but now expressed under the wild-type promoter (pBlac promoter), and to facilitate further evolution of the permuted candidate protein, we decided to change the Trc promoter of the clone with the connecting peptide NWGSE. Under the pBlac promoter context, the NWGSE clone was able to confer resistance to bacteria but only at <100 µg/ml ampicillin as compared to a resistance level of ~800 µg/ml ampicillin when expressed under the Trc promoter.

Directed evolution of the circularly permuted protein with connecting peptide NWGSE

We performed several cycles of directed evolution of the NWGSE clone expressed under the pBlac promoter. The evolutionary experiments were carried out essentially as described by Stemmer (Stemmer, 1994Go). To identify false-positive clones containing the wild-type ß-lactamase gene, we did a diagnostic PCR for the presence of the permuted gene in the antibiotic-resistant clones. This is very important to avoid working with any contaminant containing the wild-type ß-lactamase gene.

For the first cycle, we decided to select the library of variant genes at three different antibiotic concentrations (100, 400 and 600 µg/ml ampicillin). The results show that ~30% of the library is able to grow in plates containing 100 µg/ml ampicillin as compared with the control plate containing the antibiotic used to select for the cloning vector (kanamycin). Approximately 15% of the first cycle library was able to grow in plates containing 400 µg/ml ampicillin. Finally, no colonies were formed when the library was growth in plates containing 600 µg/ml ampicillin. Nucleotide sequence analysis of several clones coming from the 400 µg/ml ampicillin selection showed single and double mutations in different clones as compared with the parental sequence (Table IIGo). One of the substitutions even mapped at the NWGSE connector (Gly->Asp). We picked out four clones of this cycle to be used for the next evolutionary cycle and the produced library was selected in plates containing 800 µg/ml ampicillin. Approximately 0.01% of the second cycle library was able to grow in the selection plates. The nucleotide sequence of several clones showed a recombination of the first cycle mutations in different combinations during the second cycle (Table IIGo). From this analysis, a clear preponderance for the mutations Leu51Ile and Arg120Gly was seen. Several clones of this cycle were picked out to construct a third cycle library to be selected in 1000 µg/ml ampicillin plates. During this cycle we detected the presence of wild-type ß-lactamase in the fast growing colonies (14–16 h at 37°C). We found the permuted genes in the slow-growing colonies (visible at 2 days of incubation). Fifteen to 20 small colonies were picked out to produce the permuted genes to be used for the next evolutionary cycle. We repeated the selection step in 1000 µg/ml ampicillin plates and searched for faster growing colonies as compared with the parental ones. Only two colonies appeared, even when the expected library size plated was approximately 1x106 colony forming units. Both clones kept the above described mutations at positions 51 and 120 with new, additional substitutions. The permuted genes coming from the two colonies were used to construct the next cycle library, which was selected again in 1000 µg/ml ampicillin plates. During this fifth cycle, we observed several different colony phenotypes (only 16 colonies appeared from more than 1x106 colony forming units plated). The MIC values increased from ~400 µg/ml ampicillin for the best parent from the previous cycle to ~1000 µg/ml ampicillin for the best growing clones obtained in this evolutionary cycle. DNA sequence analysis of the permuted genes coming from the bigger colonies indicated the presence of new mutations that involved positions 104 and 111 of the ß-lactamase. It is very tempting to speculate that the modifications Glu104->Val or Glu104->Ala could be involved in improvements of the activity of the variants. Our group previously reported (Viadiu et al., 1995Go) that the change Glu104 ->Met is able to increase the activity of the wild-type ß-lactamase toward any substrate.


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Table II. Evolutionary cycles for the NWGSE-permuted protein
 
We decided to stop the evolutionary experiments at the fifth cycle and started the directed evolution of the previously described permuted protein with the GGS connector.

Directed evolution of the circularly permuted protein with connecting peptide GGS

In order to compare the evolutionary routes taken by two proteins with differences in the connecting peptide, but otherwise identical, we started directed-evolution experiments on the permuted protein with the GGS linker. It is important to note that this last protein is able to confer ampicillin resistance to bacteria but only to ~5–6 µg/ml even when expressed with a moderately strong promoter (Trc promoter) as compared with values of 800 µg/ml ampicillin resistance conferred to bacteria when the NWGSE-permuted protein is expressed under the same promoter.

Due to the low resistance levels conferred to bacteria by the GGS-permuted protein, we carried out the first evolutionary experiments of this protein expressing it from the Trc promoter.

During the first cycle of shuffling, we applied selection conditions of 10, 20 and 40 µg/ml ampicillin. Approximately 0.32% of the library was able to grow on the 10 µg/ml ampicillin plates as compared to 0.04 and 0.015% able to grow on the 20 and 40 µg/ml ampicillin plates, respectively. Sequence analysis of the permuted genes (Table IIIGo) showed a common mutation (Asn276->Asp) in different clones. MIC analysis of isolated colonies indicated a resistance level of ~200 µg/ml ampicillin for the best clones. In a second cycle of shuffling, we selected the library on 200, 400 and 800 µg/ml ampicillin plates. Two percent of the library was able to produce colonies on the 200 µg/ml ampicillin plates and a very low proportion (0.05%) of the library was able to grow on the 400 µg/ml ampicillin plates. We observed no colonies on the 800 µg/ml ampicillin plates. Sequence data for different clones showed a preference for the mutant combination Asn276Asp, Pro219Ser and His153Arg. MIC analysis of several colonies showed a resistance level in the range of 400 µg/ml ampicillin. The third cycle library was selected on plates containing 400, 800 and 1000 µg/ml ampicillin. 0.05% of the library was able to grow on the 400 µg/ml ampicillin plates and 0.01% was able to grow on the 800 µg/ml ampicillin plates. No growth was observed on the 1000 µg/ml ampicillin plates. Sequence data of several clones confirmed the above group of alterations and included the mutation Ile208->Met. MIC analysis showed that the best clones were able to grow on 1000 µg/ml ampicillin plates. For the fourth cycle, we decided to clone the library under control of the wild-type ß-lactamase promoter (pBlac). The library was selected on 100, 200 and 400 µg/ml ampicillin plates. We observed a few colonies on the 100 and 200 µg/ml ampicillin plates and no growth was observed on the 400 µg/ml ampicillin plates. Sequence data from selected clones showed a preference for the mutations at positions 276, 219, 153 and 208, described above, in addition to the change Ala280Thr. MIC analysis of several variants showed a resistance phenotype on 100 µg/ml ampicillin plates. The next cycle library was made using DNA from the best colonies from the previous, and selected on 200 and 400 µg/ml ampicillin plates. 0.1% of the library was able to grow on the 200 µg/ml ampicillin plates and no colonies were formed on the 400 µg/ml ampicillin plates. Sequence data of some clones showed the same group of mutations described for the previous cycle with some new mutations that involved positions 31, 47, 51 and 58 of ß-lactamase. MIC data showed that some variants were able to confer resistance to bacteria at levels of 300 µg/ml ampicillin. The sixth cycle of shuffling was selected on 400 and 600 µg/ml ampicillin plates. Approximately 0.35% of the library was able to grow on the 400 µg/ml ampicillin plates and 0.01% of the library grew on the 600 µg/ml ampicillin plates. Sequence data indicates the addition of new substitutions (Glu104Ala) to the group of mutations already present. All the picked clones were able to confer resistance to bacteria at 600 µg/ml ampicillin. The results from two additional evolutionary cycles showed a plateau in the MIC levels (and several new mutations were fixed) already reached in the sixth cycle, so we decided to stop the evolutionary experiments.


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Table III. Evolutionary cycles for the GGS-permuted protein
 
Specific activities of different evolved variants

In order to detect any enzymatic activity improvements during the evolutionary experiments, we decided to measure the specific activities of permuted variants using crude protein preparations from selected permuted members coming from the different evolutionary cycles. It should be noted that the results of this kind of analysis could be dependent on several factors not directly related to improvements in the activity of the protein (better expression levels, better solubility or better stability of the variants could influence favorably in a better performance in the specific activity measurements). Thus, we decided to purify the best variants (as measured by the resistance level conferred to bacteria containing those permuted genes and usually obtained at the final evolutionary cycles) coming from the two different evolutionary pathways. Ideally, one should be able to present activity data coming from the parent proteins to be compared with the best-evolved variants. Unfortunately, the low expression level (probably as a result of aggregation or stability problems) of the parent proteins has so far prevented their purification. Due to the above problems, we decided to compare the evolved variants with the parent proteins using specific activity measurements with total protein extracts.

Measurements of ß-lactamase specific activity using nitrocefin as a substrate indicate an increment of ~20-fold in the activity of a sixth evolutionary cycle clone as compared with the parental GGS-permuted protein (Table IIIGo). However, this best sixth cycle variant is still ~140-fold less active than the wild-type TEM enzyme. As discussed above, the lower specific activity of the evolved variant could be due to differences in the expression levels of the permuted protein as compared with the natural enzyme or due to poorer catalytic parameters of the permuted protein as compared with the wild-type enzyme. Determination of the catalytic parameters for the sixth cycle variant against penicillin G as a substrate showed that a combination of the above factors could be the reason for the poor activity of the evolved variant. The sixth cycle variant presents ~8% of the catalytic efficiency showed by the TEM enzyme (kcat 226.53 s-1 and Km 127.12 µM for the evolved variant as compared to kcat 782 s-1 and Km 33 µM for the wild-type enzyme).

Activity data, using penicillin G as a substrate, was previously reported for a circularly permuted class A ß-lactamase (Pieper et al., 1997Go). In that study, one variant involved new termini at the second crossover and a linker joining the old N- and C-termini (variant cp228) both of which are different from our design. The results indicate a catalytic rate (kcat) of ~0.33% the wild-type value as compared to ~30% the wild-type value showed for our purified sixth cycle GGS variant. The catalyitic efficiency (kcat/Km) decreased to 0.01% of the wild-type value for the cp228 variant and is ~8% the wild-type value for our evolved GGS variant. The different activities shown by two somehow similar permuted proteins could be due to different factors: (1) the TEM ß-lactamase fold could be more tolerant than the Staphylococcus aureus PC1 ß-lactamase to alterations in the second crossover region, resulting in a more stable and functional permuted protein; (2) the evolutionary strategy improved the stability (expression levels) and perhaps the activity of the GGS-permuted variant; (3) the slightly different position for the new N- and C-termini and the different connector peptide used for joining the old N- and C-termini could be important in the stability or function of the permuted variant; and (4) a combination of the above factors.

The specific activity measurements of clones coming from the linker randomization step showed a 7-fold improvement of specific activity for some clones with a five-residue linker as compared with the GGS-permuted protein (Table IGo). Five evolutionary cycles to the NWGSE clone resulted in a 7-fold improvement in the specific activity as compared with the parental protein and in an almost 30-fold improvement as compared with the GGS-permuted protein (Table IIGo). Unfortunately, despite expression levels similar to the sixth cycle GGS variant described above, we were unable to purify the best NWGSE variant due to problems confronted during some purification steps (which involve ion-exchange chromatography, sensitive to pI changes). We do not know if this particular NWGSE variant is sensitive to some purification conditions due to lower protein stability as compared with the sixth cycle GGS variant. We attempted to add histidine tails to either the N- or the C-terminus of the evolved NWGSE variant without success, as we observed a substantive drop of ampicillin resistance on such constructs. We did not exhaustively try modified or alternative purification procedures to get a reasonably purified NWGSE evolved variant, so, at the moment, we are unable to compare the catalytic parameters of the NWGSE-evolved variant with the wild-type enzyme values.

Analysis of the specific activity and MIC values, however, shows that we reached an upper limit at the selection step even with a putative catalytic efficiency as low as 10% of the wild-type value. This means that if we want to reach the wild-type catalytic parameters, we must attempt to increase the sensitivity of selection, perhaps by decreasing the total amount of evolved protein produced by the cell (cloning in the presence of a weaker promoter), before continuing with the evolutionary experiments.

Comparison of the evolutionary pathways taken by the NWGSE- and GGS-permuted variants

Four out of six amino acid positions that were mutated in the final evolved clones from the NWGSE route were also modified in some clones of the final evolutionary cycles from the GGS route. In some cases the targeted residue was modified to a different one due to single nucleotide changes in different positions of the codon. The most notable difference in the evolutionary routes (besides the apparent plateau reached with the GGS variant) consists of a different temporal appearance of some mutations during the evolutionary experiments: the mutation N276D and H153R appeared during the first shuffling cycle of the GGS protein as compared with the mutations N276S and H153R which appeared during the final evolutionary cycles of the NWGSE protein. The inverse happened to the change L51I which appeared during the first shuffling cycle of the NWGSE protein as compared with the mutation L51F that appeared during the final evolutionary cycles of the GGS protein. In one case, mutation E104A, the changes appeared at the final evolutionary cycles on both permuted proteins. We do not have an explanation for these facts, but due to the differences in activity (as shown by the ampicillin resistance levels and the specific activity values; see above) present in the parent permuted proteins, we expect some different problems (low activity, poor solubility, poor secretion, low stability) need to be resolved in order to increase the survival of bacteria containing the GGS-permuted protein as compared with the NWGSE protein.

Huang et al. (Huang et al., 1996Go) made a characterization of the sequence determinants of TEM ß-lactamase structure and function. In that work, the authors described a set of essential amino acid positions that do not tolerate substitutions. A subset of the putative essential residues include the positions Asp214, Val216, Ala217 and Asn276. All these residues were substituted, either by design or as a result of the evolutionary process. It is clear from our results that drastic changes, such as those needed to introduce new termini of the polypeptide chain at a crossover which is near the active site, generate a new set of constraints. The fact that substitution Asn276->Asp was found in the first evolutionary cycle of the GGS-permuted protein suggests that this mutation could be important for some improvement in the function of this permuted protein. Backcrossing experiments with the parental GGS-permuted gene should be helpful to shed light on the relative importance of the Asn276Asp mutation for the function of the evolved GGS-permuted proteins.

Conclusions and perspectives

In this work, we showed that it is possible to obtain a reasonably active circularly permuted TEM ß-lactamase that contains new N- and C-termini at the second crossover of the protein fold. This new protein has now a linear arrangement of the domain-forming residues. It is clear from our studies that successful solutions could be attained when evolving almost identical polypeptides (differences only at the junction between the old N- and C-termini) but functionally different parent molecules (the GGS connected permuted protein is practically unable to confer ampicillin resistance to bacteria as compared to the NWGSE connected permuted protein). We think it is relevant, and somehow unexpected, that the two initial constructions reached different levels of maximum activity, resolvable by our selection methods, after exhaustive random point mutagenesis and selection processes. Unfortunately, our selection system is unable to discriminate between the different factors affecting growth in the presence of the antibiotic (expression level, stability and/or secretion proficiency) and prevents us from using it to identify enzymes with wild-type levels of catalytic activity. Future work will concentrate on finding an experimental approach to increase the limit in the selection step to continue the evolutionary cycles for attaining the wild-type catalytic parameters of the evolved variants.

Much work has been published about the structure or specificity determinants of the TEM ß-lactamase (Cantu et al., 1997Go). Here, we found that residues that are invariant in the lineage (see above) are allowed to change in the permuted protein format. Clearly, the somewhat drastic change introduced by the circular permutation created a new set of constraints, illustrating the effect of jumping into a different hill of sequence-space. It could be interesting to find if the specificity determinants in a functional circularly permuted TEM ß-lactamase are similar to the ones described for the natural enzyme.


    Notes
 
1 To whom correspondence should be addressed. E-mail: joel{at}ibt.unam.mx Back


    Acknowledgments
 
We thank Paul Gaytan and Eugenio López for the oligonucleotides, Filiberto Sánchez for technical assistance, and René Hernandez and Maricela Olvera for DNA sequencing. Administrative help from Nelly Mellado is greatly appreciated. This research was supported by CONACyT grant G0030-N9608 to X.S. and PAPIIT grant IN223199 to J.O.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Ambler,R.P., Coulson,F.W., Frere,J.-M., Ghuysen,J.-M., Joris,B., Forsman,M., Levesque,R.C., Tiraby,G. and Waley,S.G. (1991) Biochem. J., 276, 269–272.[ISI][Medline]

Aurora,R., Srinivasan,R. and Rose,G.D. (1994) Nature, 264, 1126–1130.

Baird,G.S., Zacharias,D.A. and Tsien,R.Y. (1999) Proc. Natl Acad. Sci. USA, 96, 11241–11246.[Abstract/Free Full Text]

Bradford,M. (1976) Anal. Biochem., 72, 248–250.[CrossRef][ISI][Medline]

Cantu,C.,III, Huang,W. and Palzkill,T. (1997) J. Biol. Chem., 272, 29144–29150.[Abstract/Free Full Text]

Efimov,A.V. (1991) Protein Eng., 4, 245–250.[Abstract]

Goldenberg,D.P. and Creighton,T.E. (1983) J. Mol. Biol., 165, 407–413.[ISI][Medline]

Graf,R. and Schachman,H.K. (1996) Proc. Natl Acad. Sci. USA, 93, 11591–11596.[Abstract/Free Full Text]

Hennecke,J., Sebbel,P. and Glockshuber,R. (1999) J. Mol. Biol., 286, 1197–1215.[CrossRef][ISI][Medline]

Huang,W., Petrosino,J., Hirsch,M., Shenkin,P.S. and Palzkill,T. (1996) J. Mol. Biol., 258, 688–703.[CrossRef][ISI][Medline]

Humphrey,W., Dalke,A. and Schulten,K. (1996) J. Mol. Graph., 14, 33–38.[CrossRef][ISI][Medline]

Iwakura,M., Nakamura,T., Yamane,C. and Maki,K. (2000) Nature Struct. Biol., 7, 580–585.[CrossRef][ISI][Medline]

Jelsch,C., Mourey,L., Masson,J.-M. and Samama,J.-P. (1993) Proteins Struct. Funct. Genet., 16, 364–383.[ISI][Medline]

Kumar,S. and Bansal,M. (1998) Proteins Struct. Funct. Genet., 31, 460–476.[CrossRef][ISI][Medline]

Lietz,E.J., Truher,H., Kahn,D., Hokenson,M.J. and Fink,A.L. (2000) Biochemistry, 39, 4971–4981.[CrossRef][ISI][Medline]

Luger,K., Hommel,U., Herold,M., Hofsteenge,J. and Kirschner,K. (1989) Science, 243, 206–210.[ISI][Medline]

Pieper,U., Hayakawa,K., Li,Z. and Herzberg,O. (1997) Biochemistry, 36, 8767–8774.[CrossRef][ISI][Medline]

O'Callaghan,C.H., Morris,A., Kirby,S.M. and Shingler,A.H. (1972) Antimicrob. Agents Chemother., 1, 283–288.[ISI][Medline]

Stemmer,W.P.C. (1994) Nature, 370, 389–391.[CrossRef][ISI][Medline]

Viadiu,H., Osuna,J., Fink,A.L. and Soberón,X. (1995) J. Biol. Chem., 270, 781–787.[Abstract/Free Full Text]

Viguera,A.R., Blanco,F.J. and Serrano,L. (1995) J. Mol. Biol., 247, 670–681.[CrossRef][ISI][Medline]

Zaccolo,M. and Gherardi,E. (1999) J. Mol. Biol., 285, 775–783.[CrossRef][ISI][Medline]

Received June 8, 2001; revised October 2, 2001; accepted November 8, 2001.





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