Robustness of hen lysozyme monitored by random mutations

Kaori Kunichika, Yoshio Hashimoto and Taiji Imoto1

Graduate School of Pharmaceutical Science, Kyushu University,Fukuoka 812-8582, Japan


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We investigated the robustness of hen lysozyme by using random mutant libraries. Six random mutant libraries containing 1, 1.5, 2, 3, 5 and 14 amino acid mutations per hen lysozyme were systematically constructed by varying the concentrations of Mg2+ and Mn2+ on polymerase chain reaction. The mutated genes from the six libraries were cloned to a yeast expression vector and a total of 4000 clones were screened on the basis of lysis activity and ELISA employing monoclonal antibody that recognized only lysozyme with native conformation. About 80% of the clones with an average of two amino acid mutations retained active structure. Almost all clones with an average of five mutations lost active structure. On the other hand, 80% of the clones with an average of two amino acid mutations retained both gross conformation and active structure and 24% of the clones with an average of 14 amino acid mutations retained gross conformation. These results show that gross conformation is robust against mutations and so is active structure to a lesser extent.

Keywords: active structure/gross conformation/hen lysozyme/random mutagenesis/robustness


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Many approaches, such as site-directed mutagenesis (Scrutton et al., 1990Go), alanine scanning (Yu et al., 1995Go), random mutagenesis (Hecht et al., 1984Go, Alber and Wozniak, 1985Go; Matsumura and Aida, 1985Go; Lio et al., 1986Go; Makino et al., 1989Go; Joyet et al., 1992Go) and DNA shuffling (Dimitris and David, 1999Go), have been employed to analyze and improve protein structure and function. To know the robustness of protein against mutations is very important for these trials. A protein forms tertiary structure depending on its amino acid sequence and expresses its specific function (Anfinsen, 1973Go). Some tertiary structures change by one amino acid substitution and the structural changes alter the properties of the protein. On the other hand, it is said that the structure and function of proteins are stable against natural amino acid mutations (Creighton, 1993Go). Protein structure would not change so easily by a few mutations. After all, we can assume that protein in nature is stable and robust against mutations. Li et al. reported that protein structure in nature is stable against mutations with the use of a simple lattice model of protein folding (Li et al., 1996Go). Therefore, we tried to confirm the robustness of protein against mutations by applying random mutations. Directed evolution proved to be useful in enhancing performance in non-natural environments and also for obtaining new features never required by nature, provided that an efficient selection or screening method can be found to channel the enzyme evolution towards the desired properties (Arnold and Volkov, 1998Go). It mimics the process of Darwinian evolution in a test-tube combining random mutagenesis and recombination with screening or selection for enzyme variants that have the desired properties (Chirumamilla et al., 2001Go).

The purpose of this study was to prove the robustness of protein against mutations by applying random mutations. The model protein chosen here for testing robustness of protein was hen lysozyme, which is a stable protein consisting of a single polypeptide chain of 129 amino acid residues with a molecular mass of about 14.3 kDa. We prepared random libraries of lysozyme gene by error-prone polymerase chain reaction (PCR).

We showed that the mutational frequency could be regulated by altering the concentrations of Mn2+ and Mg2+. Genes from randomly mutated libraries were transferred to a yeast expression vector. Robustness against mutations was investigated by screening for the activity and the secretion from yeast monitored by enzyme-linked immunosorbent assay (ELISA). In this experiment, ELISA can check whether the protein retains tertiary structure or not. LKS103, monoclonal antibody, used in ELISA is detectable only for hen lysozyme with gross conformation because of its very high specificity for the tertiary structure. We think that mutational robustness is required for protein to adapt to environmental changes and hence this is important information.

Since there has been no report that systematically confirmed the robustness against mutations, six random libraries that possessed different frequencies of mutations were constructed and the rate of retention of activity or gross conformation was investigated.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials

Taq DNA polymerase was purchased from Roche Molecular Biochemicals (Mannheim, Germany) and restriction enzymes and T4 ligase from Takara Shuzo (Kyoto, Japan) or Fermentas MBI (Lithuania). Unfolded lysozyme was prepared by S-alkylation with (3-bromopropyl)trimethylammonium bromide (TAP-Br), following reduction of hen egg lysozyme with 2-mercaptoethanol, as described previously (Yamada et al., 1994Go). LKS1013, mouse anti hen-lysozyme IgG monoclonal antibody, was prepared by T.So and Y.Mizukami (unpublished data). Micrococcus luteus, a substrate of lysozyme, was purchased from Sigma Chemical (St. Louis, MO) or Seikagaku Kogyo (Tokyo, Japan). Dye Primer Cycle Sequencing Kits FS for DNA sequencing was purchased from PE Applied Biosystems Japan (Tokyo, Japan). Other chemicals were of analytical or biochemical grade.

Strains, plasmids

The following bacterial strains and plasmids were used: Escherichia coli RR1 (Cunningham and Wells, 1987Go), JM110 (Olphant and Struhl, 1989Go), CS3 (Hashimoto-Gotoh et al., 1993Go) and Saccharomyces cerevisiae strain AH22 (Hinnen et al., 1978Go).

Construction of random DNA library by PCR

Each PCR random library was constructed by adding various concentrations of MgCl2 and MnCl2: 100 µl of reaction mixture contained 10 mM Tris–HCl (pH 8.3), 50 mM KCl, 2.5 mM each of dNTP and 5 units of Taq DNA polymerase. PCR was carried out with a DNA thermal cycler with 10 cycles of 95°C, 45 s; 50°C, 60 s; 70°C, 90 s, followed by another 10 cycles of 95°C, 45 s; 50°C, 60 s; 70°C, 90 s + 20 s/cycle (Leung et al., 1989Go). Primers 5'-GTT TTC CCA GTC ACG ACG-3' and 5'-GGG TAT CTC TCG AGA AAA GA-3' were used to introduce XhoI and BamHI sites at the 5'- and 3'-ends of lysozyme gene, respectively.

Construction of heavily random mutant library (an average of 14 amino acid mutations)

The condition for PCR random mutagenesis (Leung et al., 1989Go) were optimized to produce an average of 14 amino acid substitutions per lysozyme molecule: 100 µl of reaction mixture contained 10 mM Tris–HCl, pH 8.3, 50 mM KCl, 6.1 mM MgCl2, 0.5 mM MnCl2, 10 mM dGTP, 2 mM dATP, 10 mM dCTP, 10 mM dTTP, 10 µM DMSO and 5 units Taq DNA polymerase. PCR was carried out with a DNA thermal cycler with 10 cycles of 95°C, 45 s; 55°C, 60 s; 72°C, 75 s, followed by another 10 cycles of 95°C, 45 s; 55°C, 60 s; 72°C, 75 s + 20 s/cycle and then 72°C for 7 min.

The resulting PCR products were extracted with ethanol, then digested simultaneously with XhoI and BamHI and ligated into pHA812 E.coli–S.cerevisiae shuttle vector with BamHI–XhoI sites. Transformation of S.cerevisiae AH22 was performed according to the spheroplast method (Hinnen et al., 1978Go). pHA812 is a derivative of pAM82 (Miyanohara et al., 1983Go), which contains PHO5 promoter and {alpha}F secretion signal sequence (Hashimoto et al., 1998Go).

DNA sequence

A randomly mutated DNA library was cloned to the M13 phage vector using E.coli JM110. The mutation frequency of each library was determined by sequencing DNA of the clones that were selected randomly. DNA sequencing was carried out with an Applied Biosystems DNA sequencer 373A and a Dye Primer Cycle Sequencing Kit (Applied Biosystems).

Screening of random mutants of lysozyme on M.luteus plates

M.luteus-overlaid plates were used for screening yeast clones which have lytic activities (Hashimoto et al., 1998Go). By observing lytic haloes, we decided whether each clone had lytic activity or not.

Determination of the amounts of secreted protein by ELISA

Determination of the amounts of secreted protein by ELISA was carried out according to the method of Kunichika et al. (Kunichika et al., 1999Go). The secreted amounts of lysozyme were determined using the ELISA system according to the method of Buchner and Rudolph (Buchner and Rudolph, 1991Go) with slight modifications. The yeast culture supernatants were used as antigen solution. We used LKS103, which is mouse anti hen-lysozyme IgG monoclonal antibody.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Control of the mutational frequency in PCR random mutagenesis by changing the concentrations of Mn2+ and Mg2+

Random mutagenesis of hen-lysozyme gene was carried out by error-prone PCR. Various protocols were tested to examine the effects of the concentrations of Mn2+ and Mg2+ on the mutation of lysozyme amplified by PCR. Increases in the concentration of Mn2+ and Mg2+ accelerated the mutational frequency (Leung et al., 1989Go; Eckert and Kunckel, 1990Go). In this experiment, we examined the correlation between the concentrations of Mn2+ and Mg2+ with mutation frequency (Figure 1Go). Consequently, it was confirmed that an increase in the concentration of Mn2+ and Mg2+ elevated the mutational frequency. Random libraries with various mutation frequencies were obtained by varying the concentrations of Mn2+ and Mg2+. In the presence of 1.5 mM Mg2+, the mutation induced at a concentration of 0.3, 0.4, 0.5 or 0.8 mM Mn2+ gave an average base substitution of 1.27, 1.78, 2.62 or 3.89 per lysozyme gene, respectively. In the presence of 0.5 mM Mn2+, the mutation induced at a concentration of 1.5, 2 or 3 mM Mg2+ gave an average base substitution of 2.62, 4.33 or 7.25, respectively. Thus, we were successful in increasing mutation frequency by increasing the concentrations of both Mn2+ and Mg2+ (Table IGo, sixth row). The mutation frequency was defined as: the number of base substitutions divided by the base number of lysozyme gene. About 10 clones from each mutant pool were selected randomly and were subjected to DNA sequencing (Table IGo). The data in Table IGo show that mutations at AT base pairs occur much more frequently than mutations at GC base pairs and that certain types of mutations such as A->T or T->A occur more frequently than others such as A->C or T->G. However, since the method with use of Taq DNA polymerase for the construction of a random library is general (Shafikhani et al., 1997Go; Wan et al., 1998Go), we employed this method. The positions of mutations were distributed uniformly throughout the lysozyme gene (data not shown).



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Fig. 1. Number of mutated base pairs against MnCl2 (A) or MgCl2 (B) concentration: (A) was carried out in the presence of 1.5 mM MgCl2 and (B) in the presence of 0.5 mM MnCl2.

 

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Table I. Mutational spectra of Taq DNA polymerase at various concentrations of Mn2+ and Mg2+
 
Construction of random mutant library by PCR

We used appropriate random libraries, which had been obtained in the above experiments and some others. The amplified PCR products were cloned to the E.coli–yeast shuttle vector, pHA812, by digestion with XhoI and BamHI. pHA812, derived from yeast expression vector pAM82 (Miyanohara et al.,1983Go), contains an addA(Smr) gene (3.6 kb) between XhoI and BamHI sites. By using the streptomycin-dependent E.coli and streptomycin-resistant gene system, only clones in which the streptomycin-resistant genes are replaced by the desired genes can be selected (Hashimoto et al., 1998Go). In the presence of 1.5 mM Mg2+, the mutation induced at a concentration of 0.3, 0.4 or 0.5 mM Mn2+ gave an average base substitution of 1.27 ± 1.85, 1.78 ± 1.48 or 2.62 ± 1.50 per lysozyme gene and amino acid mutations of 0.81 ± 1.08, 1.44 ± 1.24 or 2.08 ± 1.19, respectively. The random library in the presence of 0.5 mM Mn2+ and 2.0 mM Mg2+ had an average of 4.33 ± 2.90 for base substitutions and 3.11 ± 1.66 for amino acid mutations. In the presence of 0.8 mM Mn2+ and 6.1 mM Mg2+, it had an average of 8.36 ± 2.25 for base substitutions and 5.45 ± 1.57 for amino acid mutations. Since we wanted to know the effect of heavy mutations for lysozyme, we tried to construct the heavily mutated random library. Consequently, we constructed a random library having an average of 15.25 ± 2.50 for base substitutions and 13.75 ± 1.89 for amino acid mutations.

Screening of randomly mutated lysozyme by activity

The result of the screening of lytic haloes indicates that all clones analyzed were positive in the random library with an average of one amino acid mutation (Table IIGo). About 80% clones in an average of two amino acid mutations retained active structure. In the random library containing an average of three amino acid mutations, the rate of clones with active structure dramatically decreased to about 23%. In the library of an average of five amino acid mutations, the clones with active structure were hardly detected (Table IIGo). In Table IIGo, the rate of active clones with an average of five amino acid mutations drastically decreased to 1.6%, while the rate of active clones with an average of 14 amino acid mutations increased to 5.6%. This was an unexpected result and the screening number of clones with an average of five mutations (368) is 40% of clones with an average of 14 mutations (952). Therefore, we thought that the small size of screened clones caused this phenomenon and that this is within the error.


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Table II. Results of screening random libraries by lytic halo and ELISA
 
Lytic activity and ELISA for distinction between folded and unfolded structure

We tried to demonstrate that LKS103, mouse anti hen-lysozyme IgG monoclonal antibody, distinguishes between the folded and unfolded structures. We examined ELISA and lytic activity for native lysozyme, wild-type lysozyme secreted from yeast, mutant lysozymes (Glu35Ala, Glu35Gln, Asp66Asn and Gly71Ala) and TAP lysozyme [lysozyme S-alkylated with 3-(bromopropyl)trimethylammonium bromide] as an unfolded protein (Figure 2Go). Glu35 is a catalytic residue and Asp66 and Gly71 are in epitope sites (unpublished data). Glu35Ala and Glu35Gln mutants had no lytic activity and Asp66Asn and Gly71Ala mutants had high activities. On ELISA, TAP lysozyme had no response for LKS103. Asp66Asn and Gly71Ala mutants presented a considerable response for LKS103 and wild-type lysozyme presented a response as well as native lysozyme. These results indicated that LKS103 distinguished between the folded and unfolded structures. Also, it is also indicated that one point mutations of the epitope site moderately affected ELISA screening.



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Fig. 2. (A) Relative activities of native, wild-type and mutant lysozymes. (B) Response of mouse anti hen-lysozyme IgG monoclonal antibody, LKS103, against folded and unfolded lysozymes. Native (diamonds) and wild-type (open triangles) as folded lysozymes, Glu35Ala (closed squares) and Glu35Gln (open squares) as inactive mutant lysozymes, Asp66Asn (closed circles) and Gly71Ala (crosses) as epitope mutant lysozymes and TAP lysozyme (open circles) as unfolded lysozyme were used for antigen. The ELISA system of Buchner and Rudolph (Buchner and Rudolph, 1991Go) was applied.

 
Screening of randomly mutated lysozyme by secretion

Lysis assay can select only clones with active structure and negative clones that are selected by lysis assay contain the clones keeping a gross conformation without activity. Screening by ELISA can select only clones with gross conformation. The negative clones that were selected by lytic halo were cultured for analysis by ELISA to test whether lysozyme with gross conformation was secreted from the yeast. Consequently, {bsim}80, 50 and 24% (including activity positive clones) were secreted from yeast in random libraries containing an average of two, five and 14 amino acid mutations, respectively (Table IIGo).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mn2+ increases the frequency of mutation by decreasing the fidelity of Taq DNA polymerase (Beckman et al., 1985Go). Too high a concentration of Mn2+ disturbs the synthesis of DNA chain by Taq polymerase. The mutagenicity by Mn2+ may be directly attributable to its ability to stabilize a mispaired nucleotide on the polymerase–template complex for a long enough period to cause a substantial increase in the misinsertion frequency (Goodman et al., 1983Go). However, there is a limitation in increasing mutational frequency by increasing Mn2+ concentration. Therefore, we regulated the frequency of mutation by increasing the concentrations of Mn2+ and Mg2+ at the same time. All DNA polymerases require the presence of a divalent metal cation for activation. A number of divalent metals are mutagenic and carcinogenic and have been shown to decrease the fidelity of DNA polymerase in vitro (Mildvan and Engle, 1972Go). At high concentrations, Mg2+ was also shown to decrease the fidelity of Taq DNA polymerase (Eckert and Kunkel, 1990). We showed that the mutation frequency could be regulated by altering the concentrations of Mn2+ and Mg2+ (Figure 1Go).

We thought that the protein secreted from yeast retained gross conformation. Generally, secretory proteins are synthesized on the endoplasmic reticulum (ER) membrane and pass through the ER membrane. Structure formation, that is, protein folding, occurs in the lumen of the ER (Gething and Samstook, 1990Go, Hammond and Helenius, 1995Go). Since proteins are promptly transported from the ER to the Golgi upon folding, misfolded secretory proteins are retained in the ER by a conformational proofreading apparatus and eventually decomposed. Owing to the proofreading system, secretory proteins cannot be secreted into an extracellular compartment without maintenance of their tertiary structure in the ER (Bonifacino et al., 1991). Therefore, it is reasonable to consider that all secreted proteins retain the gross conformation. Moreover, LKS103, mouse anti hen-lysozyme IgG monoclonal antibody, has very high specificity to the tertiary structure. It is certain that all proteins detected by ELISA employed here retained gross conformation. Therefore, we could analyze the robustness for gross conformation of lysozyme.

Robustness and flexibility against environmental changes are functionally required for protein molecules (Maeshiro and Kimura, 1998Go). The evolution of natural proteins is thought to have occurred by successive fixation of individual mutations (Maynard, 1970Go). Robustness against mutations will become important information for proteins. Natural protein should be more stable and robust against mutations than we imagine from its apparent stability. Our experiment starts from this idea. We used hen lysozyme in this experiment. We constructed six random libraries containing 1, 1.5, 2, 3, 5 and 14 amino acid mutations per lysozyme molecule to investigate robustness against mutations. Here, we thought that the elevation of mutations correlates closely with magnitude of environmental fluctuation; the number of mutations increases as more environmental fluctuations occur. Martinez et al. (Martinez et al., 1996Go) demonstrated robustness of dihydrofolate reductase (DHFR) encoded by plasmid R67 isolated from the E.coli to mutagenesis by using a hypermutagenesis method. Hypermutagenesis could simulate hundreds of millions of years of evolution. DHFR retained the function even after 18 of 78 residues were mutated: this means that DHFR is robust against mutations. More than half (55%) of the position of T4 lysozyme tolerates one point substitutions to the other amino acids (Rennel et al., 1991Go). This means that proteins are fairly robust. To generalize this idea, we constructed six random libraries of lysozymes and investigated the retention of activity and gross conformation. If protein in nature is considerably robust, it might be possible to add new function to the protein whilst maintaining the original properties.

Our results show how robust lysozyme is against environmental fluctuations (mutations). First, concerning activity (active structure), all clones of the random library with an average of one amino acid mutation had lytic activity. This result indicates that one mutation did not affect the activity of lysozyme. However, mutation of a catalytic residue or a mutation of Met12 to Arg where the mutant was not secreted from yeast (Kunichika et al., 1999Go) would dramatically decrease activity or secretion. We might not have picked up such clones by accident. Such a mutant might be obtained by increasing the number of clones to be screened. About 80% of the clones with an average of two amino acid mutations retained active structure. This result shows that two amino acid mutations had little effect on activity. However, an average of three amino acid mutations drastically decreased the rate of active clones; about 20% of clones had activity. In the library containing an average of five amino acid mutations, most clones lost active structure. Concerning the gross conformation, more than 80% of the clones with an average of two amino acid mutations retained gross conformation. About 50% of the clones with an average of five amino acid mutations retained gross conformation, whereas they dramatically lost the active structure. These results indicate that gross conformation is robust against mutations and so is the active structure to a lesser extent.


    Notes
 
1 To whom correspondence should be addressed. E-mail: imoto{at}phar.kyushu-u.ac.jp Back


    Acknowledgments
 
This work was supported in part by a grant from the Rice Genome Project PR-2101, MAFF, Japan. We thank Dr Takanori So and Mr Yousuke Mizukami for providing LKS103.


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Received November 6, 2001; revised June 18, 2002; accepted July 1, 2002.





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