Arrhenius effect

As early as 1907, staphylococcal alpha-toxin was found to show the Arrhenius effect, and the mechanism of this effect has been studied by several researchers²⁸. For staphylococcal alpha-toxin, Arrhenius reported that it was inactivated by heating at 70^oC, but was reactivated by heating at 100^oC²⁸. This phenomenon has been described as the Arrhenius effect, and several workers have studied this Arrhenius effect through the investigation of staphylococcal alpha-toxin, proposing the existence of some substance which interacts with alpha-toxin at 60^oC²⁸. On the other hand, some reports indicated that active alpha-toxin aggregates at 60^oC to an insoluble, nontoxic form, whereas at higher temperatures soluble active toxin is released. Crude hemolysin of V. parahaemolyticus shows an Arrhenius effect similar to that of staphylococcal alpha-toxin^28-31. In the thermostable direct hemolysin (TDH), a major virulence factor of V. parahaemolyticus is detoxified by heating at approximately 60-70^oC, but is reactivated by additional heating above 80^oC. This paradoxical phenomenon has been shown in several strains of V. parahaemolyticus even though it still remained unexplained for almost 100 years²⁴. The previous study demonstrated that the Arrhenius effect in the TDH from V. parahaemolyticus is related to structural changes from a soluble form into a fibrils form. The native TDH (TDHn) is transformed into the nontoxic fibrils rich in β-strands by incubation at 60^oC (TDHi).

The TDHi fibrils are dissociated into unfolded conformations by further heating above 80^oC (TDHu). The rapid cooling of TDHu results in the refolding of the protein into the toxic TDHn, whereas the protein is trapped in the TDHi structure by a slow cooling of TDHu (Figure 2). TDHi, with fibrillar structure has no hemolytic activity at 37^oC, consistent with the Arrhenius effect. When TDHi fibrils are incubated above 80^oC they dissociate into unfolded states, which can further refold into toxic TDHn upon rapid cooling to 37^oC (Figure 3). This is an unusual phenomenon because the

formation of inactive protein aggregation is generally irreversible.

FIGURE 2: Effect of heat treatment on the conformation of TDH. (A) Relative hemolytic activities of TDH, measured at 37°C after various heat treatments (n = 5 per group) (B) CD spectrum of TDH at 37°C after rapid cooling(∆). The spectrum is identical to that of TDHn (dashed line). (C) CD spectrum of TDH at 37°C after slow cooling (O). The spectrum was identical to that of TDHi at 57.5°C (solid line)²⁴.

FIGURE 3: Model of heat-induced conformational change of TDH. (A) Rapid heating and cooling. (B) Slow heating and cooling²⁴.

9

Chapter 2

Global Research Goals and Design

TDH widely distributed in the strain of Grimontia hollisae and a few Vibrio species, has a variety of biological activities in animals, including hemolytic activity, cytotoxicity, and enterotoxicity in mice³². The information of physiochemical and biophysical properties of G.h-rTDH, however, have not been well reported. In this study, we aim to analyze the physiochemical and biophysical characterization of G.h-rTDH. First, the G.h-rTDH was cloned from a commercial Grimontia hollisae strain, BCRC 15890. The amino acid sequence of the cloned G.h-rTDH was compared with that of the published tdh gene, Grimontia hollisae 9041. From the results of sequence alignment, three distinct amino acid changes, i.e. Tyr53His53, Thr59Ile59, and Ser63Thr63, between tdh gene from G. hollisae BCRC 15890 and G. hollisae 9041, were observed and examined, attributed with the physiochemical and biophysical characteristics.

As mentioned previously, the TDH of V. parahaemolyticus has the ability to revert to a native form via a rapid-cooling treatment after it was unfolded at high temperatures, without any assistance of other enzymes or chemical compounds²⁴. G.h-rTDH^WT also can display the Arrhenius effect, as the protein physiochemical characterization of V. parahaemolyticus TDH has revealed.

Interestingly

the G.h-rTDHY53H/T59I/S63T

lost this Arrhenius effect. It is reasonable to speculate on the interesting points between the recovery ability and the protein structure.

The functional analysis of the amino acid residues is the most essential study to characterize the relationship between the protein function and critical amino acids.

The thermostability of G.h-rTDH might change when mutants are created. In other

words, the substitution of the amino acid residues might influence the secondary structure directly, and further affect its tertiary structure. Otherwise, it might not change the protein conformation but only permit the energy unfavorable for recovery to its native form. Briefly, by analyzing the changes in activity of G.h-rTDHY53A/T59B/S63C

(A=Y or H; B=T or I; C=S or T) mutants, the secondary structure and biophysical relationships will be better understood and elucidated.

Previous reports revealed an effect of a particular mutation on TDH from which a mutant toxin of TDH was formed from V. parahaemolyticus, R7, which has a single amino acid substitution of serine for glycine 62, constructed to show the deficiency in its hemolytic activity³³. Thus, in my experimental design, G.h-rTDHY53A/T59B/S63C

may be influencing its hemolytic activity and cytotoxicity via mutagenesis effects. In this work herein, we will study the toxic characterization of G.h-rTDH^WT and G.h-rTDHY53A/T59B/S63C

and propose to understand the mechanism of the Arrhenius effect with the strategy shown in Figure 4.

11

FIGURE 4. The flow chart describes a strategy for analyzing the relationship between protein secondary structure and biophysical characterization when the Tyr53, Thr59, or Ser63 positions of G.h-rTDH^WT were substituted into various combinations of amino acid substitutions.

Chapter 3

Materials and Methods

3.1 Bacterial strains and materials

Grimontia hollisae (BCRC 15890) was obtained in a freeze-dried form from the Culture Collection and Research Center (CCRC, Hsin-Chu, Taiwan). The bacteria were cultured in a Tryptic Soy Broth (TSB, Difco, Detroit, MI) medium, which was supplemented with 1.5% NaCl and incubated at 37°C overnight, with shaking (180 cycles/min). This strain showed the hemolytic phenomenon on agar plates containing 5% sheep erythrocytes. Phenyl Sepharose 6 Fast Flow was purchased from Amersham Pharmacia Biotech (Piscataway, NJ, USA).

3.2 Construction, expression, and purification of G.h-rTDH

^WT

protein from G. hollisae

G. hollisae were cultured in 3 mL Tryptic soy broth (TSB) medium with 3 % sodium chloride (NaCl) at 37^oC with continuous shaking for 12 h. Cultures were harvested by centrifugation at 10,000 x g for 1 min at room temperature. The supernatant was removed and the genomic DNA was extracted from the pellets using QIAamp DNA Mini Kit, following the manufacturer’s protocol (Qiagen). According to the information derived from the database from National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov) databases, the tdh gene was cloned from the Grimontia hollisae strain with two primers. The PCR conditions were similar to the general protocol previously published by our research group (Table 2)³⁴.

13

autosequencer, according to the manufacturer’s protocol (Applied Biosystems, Foster City, CA). A recombinant plasmid harboring the tdh gene was transformed into Escherichia coli BL21 (DE3) (pLys S) cells by heat shock. Transformants were cultivated at 37°C with rotary shaking in Luria-Bertani Broth (Difco) supplemented with 50 μg/mL kanamycin, and the culture was incubated for another 16 h. The cells were then harvested by centrifugation at 6,000 x g for 30 min, and resuspended in 15 mL of 20 mM Tris-HCl buffer (pH 7.0). The mixture was sonicated, and the cell debris was removed by centrifugation at 12,000 x g for 30 min at 4°C. Then the crude protein solution was loaded onto a Phenyl-Sepharose 6 Fast Flow column prequilibrated with 20 mM Tris-HCl buffer (pH 7.0) and eluted with a linear 0% to 50% ethylene glycol gradient. Fractions exhibiting G.h-rTDH activity were pooled, and added with NaCl to 200 mM concentration. The G.h-rTDH was again applied to a Phenyl-Sepharose 6 Fast Flow column with 20 mM Tris-HCl buffer (pH 7.0) containing 200 mM NaCl and then eluted with 4-fold volumes of a step gradient consisting of 200, 100, 50 and 20 mM NaCl in 20 mM Tris-HCl (pH 7.0), respectively, and sole equilibrating buffer without any salt concentration. Pure protein eluted with 20 mM Tris-HCl buffer (pH 7.0). Then, TDH protein was dialyzed against 10 mM phosphate-buffered saline buffer (PBS, pH 7.0) overnight for a hemolytic activity assay.

Table 2.

Sequences of PCR primers and PCR condition for PCR amplification of tdh gene.

Gene

G. hollisae (BCRC 15890) PCR primer sequence³⁴

tdh F 5’- ATGAAATACAGACATCT -3’

R 5’- TTATTGTTGAGATTCAC -3’

PCR Condition³⁴ Temperature (^oC) Time

Denaturation 94 ^oC 5 min

Denaturation 94 ^oC 15 sec

35 cycles

Annealing 58°C 1 min

Extension 72°C 1 min

Extension 72°C 10 min

15

3.3 Construction, expression, and purification of mutant G.h-rTDH

Y53A/T59B/S63

C protein from G. hollisae

The mutant G.h-rTDHY53A/T59B/S63C

was constructed by site-directed mutagenesis, using the recombinant plasmid harboring the tdh gene as template (tdh gene in pCR^®2.1-TOPO^®) and using two primers. The PCR condition, purification, and expression method of the mutant G.h-rTDHY53A/T59B/S63C

protein were the same as that for G.h-rTDH^WT (Table 2). The primers for construction of mutant G.h-rTDHY53A/T59B/S63C

plasmid are shown in Table 3.

Table 3. The primers sequence for construct of G.h-rTDHY53A/T59B/S63C

plasmid

tdh gene tempelate

(tdh gene in pCR^®2.1-TOPO^®)

PCR primer sequence

tdh^Y53H tdh^WT F 5’-GTAAAACGACGGCCAG-3’ (M13 forward primer)

R 5’-AAAgATgTTCACggACAATCAgTCTTCACA-3’

tdh^T59I tdh^Y53H/T59I F 5’-GTAAAACGACGGCCAG-3’ (M13 forward primer)

R 5’-TACAAAgATgTTTATggACAATCAgTCTTCACA-3’

tdh^S63T tdh^Y53H/S63T F 5’-GTAAAACGACGGCCAG-3’ (M13 forward primer)

R 5’-TACAAAgATgTTTATggACAATCAgTCTTCACA-3’

tdh^Y53H/T59I tdhY53H/T59I/S63T F 5’-GTAAAACGACGGCCAG-3’ (M13 forward primer) R 5’-ATAACgTCAggTTCTAAATggTTAACATCC-3’

tdh^Y53H/S63T tdhY53H/T59I/S63T F 5’-GTAAAACGACGGCCAG-3’ (M13 forward primer) R 5’-ggACAATCAgTCTTCACAACgTCAggTACT-3’

tdh^T59I/S63T tdh^WT F 5’-GTAAAACGACGGCCAG-3’ (M13 forward primer)

R 5’-ggACAATCAgTCTTCATAACgTCAggTACTAAA-3’

tdhY53H/T59I/S63T

tdh^Y53H F 5’-GTAAAACGACGGCCAG-3’ (M13 forward primer)

R 5’-ggACAATCAgTCTTCATAACgTCAggTACTAAA-3’

3.4 Assay of hemolytic activity

Hemolytic activity was determined on the human erythrocytes. Human erythrocytes were first washed with 100 mM PBS buffer (pH 7.0) 3 times, and then resuspended to a final concentration of 4% (v/v) in PBS buffer. For the hemolytic activity assay, 0.1 mL of 0.1% Triton X-100, which caused complete release of hemoglobin from erythrocytes and resulted absorbance change at 570 nm, was used as a positive control. Aliquots of 0.1 mL of 100 mM PBS buffer (pH 7.0) were used as negative controls. Different concentrations of the protein solution (0.1 mL) were added to the solution of human erythrocytes (0.1 mL). After incubation at 37°C for 1 h, the reaction mixtures were centrifuged at 800 x g for 5 min, and the 0.1 mL supernatant was packed. The amount of hemoglobin released from the disrupted erythrocytes was quantified by spectrophotometry on an ELISA reader at 540 nm. The 100% hemolysis activity was defined as the 570 nm absorption, with the hemoglobin released from erythrocytes treated with 0.1% Triton X-100. The equation for hemolytic activity assay is as follows:

Hemolytic activity (%) =

(protein O.D

₅₇₀

value – negative value)/(positive value – negative value) x 100

In parallel, the G.h-rTDH was subjected to 10% native polyacrylamide gel electrophoresis, and then embedded onto the agar plate containing 5% sheep erythrocytes. The blood agar plate was incubated at 37°C for an appropriate amount of time to visualize a suitable signal. In addition, G.h-rTDH proteins were also electrophoresed on a 15% SDS-PAGE and stained with Coomassie brilliant blue for

17

3.5 Analyze thermostability of the G.h-rTDH protein

The effect of temperature on hemolytic activity of purified G.h-rTDH was determined by incubating 1 μM of the purified protein in 0.1 M PBS buffer (pH 7.0) for 30 min at different temperatures (4°C, 16°C, 25°C, 30°C, 37°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C and 100°C), and then assayed for residual hemolytic activity on 4% human erythrocytes. The hemolytic activity assay method and the equation were described in section.3.4.

3.6 Compare hemolytic activity for G.h-rTDH

Y53A/T59B/S63C

The G.h-rTDHY53A/T59B/S63C

mutant was prepared at 200 μg/mL by the described xpression and purification method in section 3.3. A dilution series of protein with half concentration dilutions were incubated with 0.1 mL of 4% human erythrocytes at trypsin digestion. The in-gel trypsin digestion experiment, excision of protein bands from polyacrylamide gels and the gel particles were prepared for in-gel digestion and washed with 50 μL wash buffer (10mM NH₄HCO₃, 50% ACN) for 15 min. All remaining liquid was removed, and 100 μL ACN was added to cover the gel particles for 20 min. When the gels shrink and stick together, 3 μL of trypsin (20 ng/mL) was added to the gel and incubated for 1 h at 4^oC, and then incubated at 37^oC overnight.

The reaction was stopped with 1% TFA, and 10 min sonication, supernatant recovered, then the sample was directly mixed with MALDI matrix (CHCA, 20 mg/mL in 50%

ACN, 0.1% TFA), and analyzed using an autoflex III (BRUKER). After a default calibration, all MS spectra were recorded in positive reflector mode within a mass range of m/z 500–4000. For an initial MS scan, 4 subspectra with 200 shots per subspectrum were accumulated for each spot using a random search pattern. Spectral peaks were included in the acquisition list for the MS/ MS run of the result-dependent experiment, if they met threshold criteria (S/N above 6). For MS/MS experiments, 2,000 shots per spectrum were accumulated. Subsequently, all acquired MS/MS spectra were searched against the Swiss-Prot database using the MASCOT search engine (biotools, v3.1). Search parameters for peptide and MS/MS mass tolerance were 100 ppm and 100 ppm, respectively, with the allowance for one missed cleavage made from the trypsin digest. The search mode was carried out to identify the variable modification of oxidation (M), and Carboxymethyl (C) groups at the C terminus.

Proteins were identified by PMF and MS/MS with MASCOT, which corresponds to p<0.05.

3.8 Difference scanning calorimetry (DSC)

DSC measurements were performed using the DSC-Q10 (TA instruments). The DSC-Q10 was run without feedback and 10 min equilibration times at 25^oC were used as previously described. The protein was scanned from 25^oC to 95^oC at a heating rate of 0.5 ^oC/min. A pan containing 10 mM Kpi buffer, pH 7.0 was used as a reference.

The sample and reference cells of optical operational volume of 0.5 mL were used.

Protein samples were concentrated to 0.36 mg/mL in 10 mM Kpi buffer (K2HPO4, pH 7.0). DSC data were corrected for instrument baselines and normalized for scan rate and protein concentration. Data obtained for TDH protein were analyzed with the TA

19

3.9 Circular dichroism (CD) spectroscopy

Circular dichroism (CD) spectra were recorded with a J-815 spectropolarimeter (JASCO, Tokyo, Japan) equipped with a thermoelectric temperature controller. Data were processed with software provided by JASCO. Measurements were taken in a quartz cuvette with a path length of 1 mm, and scanned in the interval of 0.2 nm at a rate of 50 nm/min. The data from 6 individual replicates were averaged. The protein concentration was 0.18 mg/mL in 10 mM Kpi buffer (pH 7.0) for the measurement of far-UV (190-250 nm) CD spectrum. The experimental temperatures were 37°C, 50-60°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, and its Tm value, respectively (the Tm

value was determined from DSC data). Before measurement, the sample was pre-equilibrated at each experimental temperature for 5 min. The mean residue ellipticity, [Θ], was calculated by using the following relationship:

[Θ] was expressed in degrees squared centimeters per decimole.

3.10 Cell line

The AGS cell line (BCRC 60102) is a human stomach adenocarcinoma cell taken from a 54-year-old female Caucasian. Cells were maintained in RPMI Nutrient Mixture (Gibco) supplemented with fetal calf serum (10%, v/v, Gibco) and penicillin streptomycin (1%, v/v, Gibco). Cells were incubated at 37°C in an incubator of 5%

CO₂ in air. Every 2 to 3 days the culture medium was renewed, and doubling time of this cell line was 20 h.

3.11 Morphology examination

The AGS cells were first cultured in a 6-well plate overnight. Before examination, 100 mM PBS buffer (pH7.0) was used to wash the cells twice, then cells were mixed with 10 g/mL G.h-rTDH in RPMI complete medium for 30 min at 26^oC. Subsequently, the images of the cell morphology were recorded with the 200x magnification by an Olympus digtal camera.

3.12 MTT assay

For cytoviability, the MTT assay has been widely used as a colorimetric assay for measuring the activity of mitochondrial dehydrogenase that reduce MTT, a tetrazolium dye: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, substrate to form formazan, which is generated only by living cells but not dead cells.

In this assay, AGS cells were inoculated in 200 uL of complete growth medium at a concentration of 2 x 10⁴ cells in a 96-well microtiter plates. Plates were incubated for 24 h at 37^oC in an atmosphere of 5% CO2 in air. Different concentrations of G.h-rTDH were added and treated for 1 h before the supernatant was removed via a centrifugation separation. Fresh MTT solution (5 mg/mL) was dissolved in PBS and filtered by a 0.22 filter, and was diluted 10-fold in a complete medium. A 100 ul aliquot of the above solution was added to each well. Plates were wrapped by the aluminum foil and incubated for a further 5 h at 37 ^oC. MTT was then removed from the wells, and the formazan crystals were dissolved in 200 ul of Dimethyl sulfoxide

21

experiments.

3.13 Thioflavin T florescence assay

Thioflavin T (ThT) is a benzothiazole compound that possesses the light-emitting component of leuciferin. It can be utilized to visualize the amyloid beta content of protein in the solution. ThT binds to amyloid fibrils and the florescence intensity can identify the fibril content by florescence spectra. A 10 g sample of G.h-rTDH protein was respectively and sequentially heated to 37^oC, 70^oC and 90^oC for 15 min and then subjected to rapid cooling in ice water for 15 min.

Before measuring the florescence of the fibril content in the solution, ThT and Tris-HCl buffer were premixed. The ThT/Tris solution was as described: ThT powder was dissolved in distilled water and the final concentration was 1 mg/mL (3.14 mM).

Then, 1.6

l of ThT solution was added to 1 mL 50mM Tris-HCl, pH7.0 buffer.

Before measurement, an aliquot of 200

l ThT/Tris solution was mixed with

thermally pre-treated protein, the fiber forming protein containing beta sheets became immediately bound to ThT. The florescence of Thioflain T was measured at 460-600 nm when excited at 450 nm using fluorescence spectrophotometry (Hitachi, F-7000).

The Thioflavin T was obtained from Sigma (St. Louis, MO).

Chapter 4 Results

4.1 Cloning, sequence analysis and identification of the G. hollisae tdh gene

The nucleic acid sequences of tdh gene from various Vibrio species were aligned and analyzed to find the highly conserved sequences. The primers for the conserved gene were thus designed, and used in the polymerase chain reaction (PCR) to amplify the putative tdh gene from G. hollisae BCRC 15890 genomic DNA. The construct plasmid carry out tdh-mutated gene was obtained from the lab. The PCR amplified G.

hollisae tdh gene is 570-bp in size and encodes a polypeptide of 189 amino acids involving a signal peptide with 24 amino acids in the N-terminal region, and a mature protein of 165 residues, with a predicted molecular mass of 18,616.9 Da. Notably, three distinct amino acid changes, i.e. Tyr53His53, Thr59Ile59, and Ser63Thr63, were observed from the sequence alignment between the PCR amplified tdh gene from G. hollisae BCRC 15890 genomic DNA (assigned as G.h-rTDH^WT) and the historically published tdh gene from G. hollisae 9041 in the NCBI data bank (assigned as G.h-rTDHY53H/T59I/S63T

). Amino acids sequence alignment between the two tdh genes is shown in Figure 5.

23

FIGURE 5. The sequence alignment of putative tdh genes between the PCR amplicon tdh gene from G. hollisae BCRC 15890 genomic DNA (assigned as G.h-rTDH^WT) and the historically published tdh gene, from G. hollisae 9041 as described in the NCBI data bank (assigned as G.h-rTDHY53H/T59I/S63T

). The sequence of the signal peptide with 24 amino acids in the N-terminal is shown in red. The distinct amino acid change between G.h-rTDH^WT and G.h-rTDHY53H/T59I/S63T

are red boxed.

4.2 Expression, purification, determination and hemolytic activity of G..h-rTDH

^WT

The wild type G. hollisae tdh gene G.h-rTDH^WT was cloned into the plasmid pCR^®2.1-TOPO^® and subsequently transformed into Escherichia coli BL21(DE3)(pLysS) cells for protein expression. Following the incubation for 16 h at 37^oC, the harvested cells were sonicated for the expressed protein purification using a Phenyl-Sepharose 6 Fast Flow column. After the first round of chromatographic purification for the separation of impurities from crude extraction, the homogenous protein with a molecular mass of approximately 22 kDa, as resolved by 15%

SDS-PAGE was collected from the subsequent purification on the same Phenyl-Sepharose 6 Fast Flow column (Figure 6). A single band at approximately 90

kDa was observed by 10% native-PAGE, and the hemolytic activity of this protein band suggested that it is an active tetrameric protein under physiological conditions (Figure 6). MALDI-TOF MS spectrum of peptide mapping via a trypsin digestion further confirmed the identity of G. hollisae TDH.

FIGURE 6. Purification and characterization of the G.h-rTDH^WT protein from G.

hollisae. (A) The crude protein without tdh gene insertion in a pCR^®2.1^®-TOPO plasmid was obtained in the BL21(DE3)pLysS strain (lane 1). The crude protein containing the expressed G.h-rTDH^WT in the BL21(DE3)pLysS strain was also included (lane 2). The homogenous protein with a molecular mass of ~22 kDa was obtained via two Phenyl Sepharose 6 Fast Flow chromatography runs (lane 3 and lane 4). (B) Native-PAGE of purified G.h-rTDH^WT, with a molecular mass of ~90 kDa. (C) Hemolytic activity of G.h-rTDH^WT.

25

4.3 Identification of G.h-rTDH

^WT

by MALDI-TOF-TOF MS spectrometry

The purified protein was then subjected to a MALDI-TOF MS spectrometry for the internal amino acid determination to confirm the identity of purified protein. The SDS-PAGE band corresponding to G.h-rTDH^WT was first applied to an in-gel trypsin digestion as described by Rosenfeld et al.³⁵. The cutting sites of trypsin are lysine (Lys, K) and arginine (Arg, R), respectively. After digestion, the resulting peptide mixtures were analyzed by MALDI-TOF MS. Among these peptide fragments shown in Figure 7, via a database search for the mass spectrum of the peptide and its fragment ions, the tandem mass spectrum of mono-charged precursor was observed at m/z 1024.543, 1365.788, 1690.949, 2346.302, 2953.530. Following the analysis of the highest signal by MS/MS, the sequence of this fragmental was determined to be

35VSDFWTNR⁴² of G. hollisae TDH.

FIGURE 7. (A) MALDI-TOF-TOF MS spectrum and peptide mapping of G.h-rTDH protein. (B) Tandem mass spectrum of a signal charged tryptic peptide at m/z 1024.543, was deduced from the mass differences in the y-fragment ion series and partial b-ion and a-ion.

27

4.4 Expression, purification, and hemolytic activity determination of mutated G.h-rTDH

Y53A/T59B/S63C

In order to understand the role of these distinct amino acid changes between the

In order to understand the role of these distinct amino acid changes between the

在文檔中 探討霍氏格里蒙菌中熱穩定性溶血素之原始型與突變型生化活性與生物物理特性的差異 (頁 17-0)