Bachelor Degree Project

Uppsala University

Department of Chemistry—BMC

Bachelor Degree Project

Production and purification of β-ureidopropionase

variants for structural and functional analysis

Jingxuan Tai

Supervised by

Doreen Dobritzsch, Associate Professor

Dirk Maurer, PhD student

Contents

Abstract

1.Introduction

1.1 Reductive pyrimidine degradation and β-ureidopropionase ... 5

1.2 Aim of this project ... 8

2. Materials and methods

2.1 Preparation of media, solutions and gels ... 9

2.2 Cell growth and gene expression ... 10

2.3 Cell harvest and lysis ... 10

2.4 Purification steps ... 11

2.4.1 IMAC ... 11

2.4.2 Desalting, tag-cleavage and Reverse IMAC ... 11

2.4.3 Ion exchange chromatography... 12

2.5 Protein analysis ... 13

2.5.1 SDS-PAGE and Native-PAGE ... 13

2.5.2 Determination of protein concentration ... 13

2.5.3 Light scatting ... 13

2.5.4 Activity measurement ... 13

2.5.5 Crystallization... 15

3. Results and Discussion

3.1 Expression and Purification ... 15

4. Appendix

4.1 List of abbreviations, formulas and symbols ... 25

4.2 Dynamic light scattering report ... 27

4.3 Crystallization conditions ... 29

5. Acknowledgement

Abstract

1. Introduction

1.1 Reductive pyrimidine degradation and β-ureidopropionase

Pyrimidines play an important role in living cells. The most commonly known pyrimidines are the pyrimidine nucleotide bases of DNA and RNA, thymine, cytosine and uracil. The reductive pathway for degradation of these pyrimidines helps cells to degrade superfluous pyrimidines and keep a balance of pyrimidine nucleotide concentrations. The reductive pyrimidine degradation is achieved through three subsequent enzyme-catalyzed reactions[1,2]_{（see Figure 1, this figure was adapted from}

reference3）. β-ureidopropionase (EC 3.5.1.6), the third enzyme involved in this process, catalyzes the hydrolysis of N-carbamoyl-beta-alanine to β-alanine [3]_{. Also,}

the pyrimidine degradation pathway is the only source of β-alanine in mammals [4]_.

β-ureidopropionase from different eukaryotic species such as bovine, rat and

Drosophila melanogaster [5,6,7,8]_{, has been purified and characterized.}

β-ureidopropionase from Drosophila melanogaster (DmβUP) shares 64% identity with human β-ureidopropionase and the structure of DmβUP has already been determined. The crystallization result of β-ureidopropionase from Drosophila

melanogaster [9] _{shows us that it exists as a mixture of different oligomer states, such}

as dimers, tetramers and even higher oligomers. These higher oligomers forms by the hydrophilic or polar interaction of assembled dimer units’ surface (see Figure 2, this figure was adapted from reference 7) and we hope that they can be stabilized by specific changes in the amino acid at the dimer-dimer interface.

Figure 2: Schematic representation of DmβUP homo-octamer assembly. The dimer-dimer interfaces were colored in magenta and green.

To stabilize a particular oligomeric state of the enzyme for subsequent crystallographic studies, site-directed mutagenesis was used to create the mutations we purified in this project: R130D/S208R and S208C.

The position of R130 and S208 can be seen in Figure 3. These figures are created with a homology model of the human enzymes, which was created using the structure of the Drosophila enzyme. From the structure of β-ureidopropionase we can see that both the R130 and S208 residues have the interaction with another dimer in the dimer-dimer interface.

R130D and S208R are designed to eliminate the hydrogen bonding interactions and ion-ion interactions, also to create steric clashes. Therefore, the interface between dimers is supposed to be prevented so that R130D/S208R probably exists as dimers.

(a)

(b)

Figure 3: Residues S208 (a) and R130 (b) on the interface between dimeric units D and E

1.2 Aim of this project

The aim of this proposed project can be divided into two parts:

The first aim was to over-express and purify two mutant variants of β-ureidopropionase: R130D/S208R and S208C: using E. coli strain BL-21 (DE3) cells as competent cells and vector pET 151/D-TOPO encoding the gene of β-ureidopropionase should be transferred into the cells. Also use Immobilized metal affinity chromatography and anion exchange chromatography to purify β-ureidopropionase.

electrophoresis, Static light scattering and dynamic light scattering to study the structural feature of this two mutation compared with wild type β-ureidopropionase. Crystallization of mutations, which can give further insights about the structure of β-ureidopropionase, could also be attempted by using commercial sparse matrix crystallization screens.

2. Materials and methods

2.1 Preparation of media, solutions and gels

LB media: 1% (w/v) Peptone; 1% (w/v) NaCl; 0.5% (w/v) Yeast extract

TB media: 1.2% (w/v) Peptone; 2.4% (w/v) Yeast extract; 0.5% (v/v) Glycerol; 100mM NH4Cl; 20mM MgSO4; 200mM KH2PO4

Antibiotics: 100mg/ml Ampicillin; 50mg/ml Kanamycin; both are 1:1000 diluted when used

Buffer A: 50mM NaCl; 20mM HEPES; pH 7.4

Buffer B: 800mM NaCl; 20mM HEPES; pH 7.4

SDS-PAGE Running Buffer: 2.5mM Tris; 192mM Glycine; 0.1% (w/v) SDS; pH 8.3

SDS-PAGE Sample Buffer: 240mM Tris-HCl pH 6.8; 40% (v/v) glycerol; 8% (w/v) SDS; 0.04 % (w/v) bromophenol blue; 5 % (v/v) -mercaptoethanol

Staining solution: 0.1% (w/v) Commassie Blue R250 in 10% (v/v) HAc; 40% (v/v) MeOH; 50% (v/v) H2O

SDS-PAGE gels: see Table 1

Table 1: Recipe for SDS-PAGE gels

Gel (10% SDS) Separation Gel (1x) Stacking Gel (1x)

Milli-Q 2ml 1.5ml 1.5M Tris-HCl, pH8.8 1.25ml - 0.5M Tris-HCl, pH6.8 - 0.625ml 10% (w/v) SDS 50μl 25μl Acrylamide (30% w/v) 1.67ml 0.33ml 10% (w/v) APS 25μl 12.5μl TEMED 5μl 2.5μl Total volume 5ml 2.5ml

2.2 Cell growth and gene expression

In this experiment, we used E. coli strain BL-21 (DE3) cells as competent cells. The vectors pET 151/D-TOPO_R130D/S208R and pET 151/D-TOPO_S208C were already electro-transferred into the BL-21 cells, which means that adding arabinose can cause the over-expression of β-ureidopropionase mutants. Additionally, vector pREP4 containing the GroEL/ES gene sequence was also already transferred into the cells. Increasing the concentration of IPTG can achieve the co-expression of GroEL/ES, which belongs to the chaperonin family of molecular chaperones.

4 µl BL-21 cells were incubated in 4 ml LB media containing 100mg/L ampicillin and 50mg/L kanamycin at 37 °C with shaking overnight. Three separate cultures were prepared. Then for scale up, 50 µl of the last day´s culture was used to inoculate 50 ml LB media containing 100mg/L ampicillin and 50mg/L kanamycin that were incubated at 37 °C overnight.

Subsequently, cells were incubated in 500 ml TB medium at 37 °C until the OD600nm

reached 0.4~0.6. 1mM IPTG and arabinose were added for inducing the cell before the overnight shaking at room temperature. However, when S208C expressing cells were grown for the second time, IPTG was not added to prevent co-expression of chaperones. This was done to see whether it would lead to fewer contaminant proteins being co-purified with β-ureidopropionase without co-expressing chaperones.

2.3 Cell harvest and lysis

Cell lysis was usually performed by addition of lysis buffer (buffer A with additional 10μg/ml DNase, 50μg/ml Lysozyme) to a final volume of 200 ml for suspending 20 g cells. The sample was then homogenized with the homogenizer Ultra-turrax T45 (IKA WERK), lysed in a sonicator operating at 1.7 kbar (8×20 s) and centrifuged at 16000×g and 4°C for 60min. The supernatant was kept for further purification. 10μl supernatant were analyzed by SDS-PAGE. The proteins were always kept in cold room or icebox from now.

2.4 Purification steps

2.4.1 IMAC

The first step we chose for purification was immobilized metal affinity chromatography (IMAC). IMAC is a technology taking advantage of the affinity of metal ions and organic compounds to purify proteins with His tags. First, nickel is bound to an agarose bead by chelation using nitrilotriacetic acid (NTA). NTA binds Ni2+ _{ions via four}

coordination sites. Since the β-ureidopropionase variants contain a designed His tag, they will bind to the Ni2+ _{and be retained on the column. The low initial imidazole}

concentration helps to prevent nonspecific binding of endogenous proteins that have histidine clusters [10]_.

The lysate supernatant was mixed with Ni2+_{-NTA beads on a shaker for 1 h, and then}

the suspension was transferred to an empty PD-10 column (from GE Healthcare). After that, the column was washed with 30 ml Buffer A containing 60mM imidazole to wash out nonspecific binding proteins. The β-ureidopropionase protein was eluted by washing the column with 30 ml Buffer A containing 600mM imidazole. Due to the competition with the high concentration of imidazole, the protein will loose the high affinity with the Ni2+ _{and be eluted from the column. 10μl of solutions after washing}

and elution steps were also taken as the SDS-PAGE samples.

2.4.2 Desalting, tag-cleavage and Reverse IMAC

taken as the SDS-PAGE samples.

Next, tag-cleavage by an enzyme called TEV protease was performed in preparation of a reverse IMAC step in which we do not want our protein to bind to the Ni2+ _beads.

TEV protease was added to a final concentration of 0.1mg/ml and β-mercaptoethanol was added to a final concentration of 5mM to the desalted sample, which was then incubated overnight.

After the overnight reaction, imidazole was added to a final concentration of 60mM, and the protein solution was loaded onto the Ni-NTA column again. 30 ml of Buffer A containing 60mM imidazole were then applied, and all flow-through was collected. Then the proteins binding to the resin were eluted by washing the column with 20 ml Buffer A containing 1M imidazole. 10μl of solutions from Reverse-IMAC wash and elution fractions were taken as SDS-PAGE samples. Usually, the previously described desalting step was repeated after the reverse-IMAC.

2.4.3 Ion exchange chromatography

An anion exchange column (5ml HiTrap Q HP from GE Health Care) connected to an Äkta explorer system was used in this step of purification. Ion exchange chromatography is a chromatography process that separates ions and polar molecules based on their affinity to the ion exchanger. For example, if the pH of the buffer solution is higher than the isoelectric point of the protein of interest, the protein will be negatively charged and interact with an anion exchange column, while molecules with positive or no charge will flow through. A salt gradient is then used for elution; as the salt concentration in the buffer increases, molecules interacting weakest with the anion exchanger will elute first. As a result, proteins can be separated by their isoelectric point after passing an ion exchange column.

In our experiment, we applied a stepwise gradient of Buffer A (50mM NaCl; 20mM HEPES; PH 7.4) and Buffer B (800mM NaCl; 20mM HEPES; PH 7.4), with steps at 0%, 13%, 35%, 100% Buffer B, to separate the aimed β-ureidopropionase variants from contaminants. Buffer A was used to load the sample onto the column. The concentration of salt was always increased just after the flow-through or respective elution peaks, detected via absorbance at 280 nm, were fully eluted fractions were collected and analyzed by SDS-PAGE to identify these peaks. After the fractions containing the purified β-ureidopropionase were identified, they were concentrated, aliquoted, frozen in liquid nitrogen and stored at -80°C for further analysis.

2.5 Protein analysis

2.5.1 SDS-PAGE and Native-PAGE

Polyacrylamide gel electrophoresis (PAGE) is a wide-used technology in separating proteins and nucleic acids. Native Page keeps the oligomeric form intact and will show a band on the gel that is representative of the size of the physiologically important protein form, whereas SDS PAGE will denature and separate the oligomeric form into its monomers and will show bands that are representative of their molecular weights. SDS PAGE can be used to access the purity of and identify the protein [11,12]_.

For SDS-PAGE, 10μl of protein sample was mixed with 20μl of bromophenol-containing sample buffer and then heated at 95°C for 10 min. The samples were loaded to the gel and run at 120V while passing the stacking gel and at 200V for the separation gel. The separation gel was stained in hot staining solution for 20 minutes with shaking, and then distained by shaking in distaining solution at room temperature for 1 h.

For the Native-PAGE, no SDS was present in the gel and sample buffer, and the samples were kept on ice before loading and not heated. The gel was run in the cold room at 150V for 3 hours.

2.5.2 Determination of protein concentration

Due to the absorbance of Trp, Tyr and Cys at 280 nm, proteins always have a special absorbance at 280 nm. Based on Lambert-Beers law A=ε·l·c the concentration can be determined by measuring its absorbance at 280 nm. (In this experiment, ε(β-ureidopropionase) =56630 M-1_cm-1_{and path length l= 1 cm).}

2.5.3 Light scatting

Both static light scattering (SLS) and dynamic light scattering (DLS) experiments were performed for the two purified enzyme variants. Static light scattering is a physical chemistry technique that measures the intensity of the scattered light to obtain the average molecular weight Mw of a macromolecule like a polymer or a protein in solution. Dynamic light scattering can also measure polydispersity and aggregation properties of proteins.

The protein was diluted to 2 mg/ml for SLS, and 1 mg/ml or 5 mg/ml for DLS.

The principle of the activity assay is shown in Figure 4.

Figure 4: Principle of the activity assay

The activity of the β-ureidopropionase variants R130D/S208R and S208C was assayed in quadruplets, with wild-type β-ureidopropionase as positive and buffer as negative control. To start the reaction, 10mM 3-ureidopropionic acid was mixed with 0.2mg/ml enzyme (or buffer A) and left to react for 30 min at room temperature. Then 5μl of 5M NaOH was added to stop the reactions followed by addition of 230μl assay reagent (1.5mM Ortho-phthalaldehyde, 1.5mM Na2SO3, 0.1mM EDTA and 280mM Na-borate

Fluorescence was measured at 460 nm after excitation at 340 nm.

2.5.5 Crystallization

By using commercial sparse matrix crystallization screens (Morpheus Ⅱ from Molecular Dimensions Company), eight kinds of different mixes of additives, at threedifferent pH, and four precipitant mixes (detail information see appendix) were tried to crystallize the R130D/S208R variant. All crystallization plates were incubated at 20°C.

3. Results and Discussion

3.1 Expression and Purification

First R130D/S208R purification

Figure 5: SDS-PAGE result of the first R130D/S208R purification, IMAC step

1: Marker, 2: IMAC flow through, 3: IMAC 60mM imidazole wash, 4: IMAC 600mM imidazole elution, 5: Reverse IMAC flow through, 6: Reverse IMAC 1M imidazole elution fraction, 7: concentrated β-ureidopropionase

Figure 6: SDS-PAGE result for the anion exchange chromatography step of the first

purification of R130D/S208R.

1: Marker, 2: IEC 13% Buffer B elution, 3: IEC 35% Buffer B elution, 4: IEC 100% Buffer B elution.

Second R130D/S208R purification

Figure 7: SDS-PAGE result of the second IMAC purification of R130D/S208R

1: Marker, 2: Reverse IMAC 1M imidazole elution, 3: Reverse IMAC flow through, 4: PD-10 elution, 5: PD-10 flow through, 6: IMAC 600mM imidazole elution, 7: IMAC 60mM imidazole wash, 8: Lysate

Figure 8: SDS-PAGE result of the second IEC purification of R130D/S208R and first IEC

purification of S208C

1: Marker, 2: R130D/S208R IEC 13% Buffer B elution, 3: S208C IEC 13% Buffer B elution, 4: S208C IEC 35% Buffer B elution, 5: S208C IEC 100% Buffer B elution

First S208C purification

buffer B. SDS-PAGE analysis indicated sufficient purity for the S208C fraction eluted with 13% Buffer B (see Figure 8).

Figure 9: SDS-PAGE result of S208C first time

1: Marker, 2: IMAC 60mM imidazole wash, 3: IMAC 600mM imidazole elution, 4:

Concentrated elution, 5: PD-10 flow through, 6: PD-10 elution, 7: Reverse IMAC flow through, 8: Reverse IMAC 1M imidazole elution, 9: Concentrated protein

Second S208C purification

In the second expression and purification experiment for S208C we did not add IPTG at induction point, which prevents co-expression of chaperones. The faintness of the bands in the SDS-PAGE (see Figure 10 and Figure 11) following the purification indicates that as a result the expression yields were very low.

Figure 10: SDS-PAGE result of the second S208C purification

1: Marker, 2: IMAC 60mM imidazole wash, 3: IMAC 600mM imidazole elution, 4:

Figure 11: SDS-PAGE result for S208C second time IEC.

1: Marker, 2: IEC 13% Buffer B elution, 3: IEC 35% Buffer B elution, 4: IEC 100% Buffer B elution.

3.2 Yield analysis

The yield of cells and β-ureidopropionase can be seen in Table 2. The masses of the purified β-ureidopropionase variants were calculated from the absorbance at 280 nm.

Table 2: Yield of production and purification

Volume of TB media (L) Mass of cells collected (g) Mass of protein purified (mg) R130D/S208R

first expression & purification 6 46.6 14.3 R130D/S208R second expression & purification 3 25.8 1.7 S208C first expression &

purification 3 27.9 1.2 S208C second expression & purification 3 25.1 0.4

expression experiment for R130D/S208R and the first S208C expression resulted in a relatively low yield of protein, which was probably because the columns are old ones so there may be some contaminants in the column or the affinity of the column was not good causing less protein bound to the column, also the chaperones made it even hard to purified. So, we used new PD-10 columns and new concentrators for the next purification. Also, we tried not to add IPTG in the second experiment for the S208C mutant, to test whether suppression of co-expression of chaperones would eliminate chaperones being co-purified with this variant. However, the protein concentration was very low even from the beginning of purification. So, we can conclude that chaperone co-expression plays an important role for obtaining high yields of the β-ureidopropionase variants.

3.3 Structural analysis

3.3.1 Native-PAGE

Figure 12: Native-PAGE result of wild type β-ureidopropionase and mutant variants. 1: S208C, 2: R130D/S208R, 3: C233A, 4: K132L, 5: S208R, 6: R130D, 7: Wild type

β-ureidopropionase

3.3.2 Light scattering

The molecular weight of a β-ureidopropionase monomer is 43kDa, so the result from light scattering (see Table 3) tells us that R130D/S208R exists mainly as dimers and S208C is a mixture of monomers and dimers. This result agrees with the Native PAGE result. As for the β-ureidopropionase wild type, we can know that their molecular weight is much higher so they mainly exist between dimers and tetramers. As a conclusion, for the mutant R130D/S208R, we succeeded in eliminating the targeted hydrogen bonds at the dimerization interface. For the mutant S208C, we expect it exists as high oligomeric forms but there was an unexpected that one hydrogen bond would have such a pronounced effect on the quaternary structure of β-ureidopropionase.

Table 3: Molecular weight and polydispersity results of light scattering (complete report can be seen in the Appendix 4.2)

R130D/S208R S208C Wild type

SLS Mw (kDa) 91.23 64.62 —

DLS Mw (kDa) 77.15 64.64 109.88

DLS Polydispersity (%) 29.3 57.4 67.8

3.3.3 Crystallization

After incubating at 20 °C for 7 days, we found star-shape crystals in the plates (see

Figure 13). These star crystals may be 2 or 3 crystals connected with each other or

point for further optimization of the identified conditions, and these crystals can also be used as seeds for further crystallizations.

Figure 13: Crystal of β-ureidopropionase

There were crystals found in 3 different wells of the Morpheus® screen: A3.3 (Figure 9), G2.3 and A7.3. Table 4 shows the conditions of these 3 wells and detailed

information of composition can be seen in Appendix 4.3.

Table 4: Conditions producing β-ureidopropionase crystals.

No. Additives Precipitant PH

Protein concentration

(mg/ml)

A3.3 LiNaK Precipitant Mix 7 6.5 6

G2.3 LiNaK Precipitant Mix 6 6.5 6

3.4 Activity measurement

The activity test result can be seen in Table 5 (protein final concentration used in the assay was 35μg/ml). From enzyme activity tests performed in quadruplet, we can conclude that both R130D/S208R and S208C are inactive. This result agrees with the hypothesis that assembly of at least two dimers to a tetramer is required to enable the ordering of active site loops located at the dimer-dimer interface for the active site to be fully formed.

Table 5: Activity tests results of wild type and two mutants (μmol formed

product/min)

Test Wild type R130D/S208R S208C

1 1.626 0.275 0.018

2 3.488 0.155 0.048

3 2.299 0.113 0.073

4 1.434 0.096 0.001

3.5 Conclusion

The propose of this project was to over-express and purify two mutants of β-ureidopropionase, R130D/S208R and S208C, to analyze their quaternary structure and activity, and also to identify conditions for crystallization of mutant R130D/S208R.

The development of this project can be concluded as a success. In this project, two variants of β-ureidopropionase, carrying the mutants R130D/S208R and S208C, were both successfully produced, purified and analyzed. In total, we got 16.0mg pure R130D/S208R protein and 1.6mg pure S208C protein. According to the enzyme activity test, we found that both these two mutant enzymes are inactive, which corresponds well to our hypothesis that assembly of at least two dimers to a tetramer is required to enable the ordering of active site loops located at the dimer-dimer interface. Only when these loops are ordered is the active site fully formed. Combined with Native-PAGE and light scattering result, we can conclude that they exist mainly in dimers. And hence we succeeded with stabilizing a particular oligomeric state of the enzyme for subsequent crystallographic studies. Also, R130D/S208R crystals were obtained from 3 different conditions, so we can establish conditions which can be helpful with the crystallization for β-ureidopropionase.

With respect to method validation and optimization, we found that:

• Induction of chaperone co-production by IPTG plays an important role in β-ureidopropionase protein expression, as there β-ureidopropionase yields were much lower in the cells to which no IPTG was added.

• Adding additional 5mM β-mercaptoethanol in buffers will help the purification of S208C. That is because β-mercaptoethanol is a reduction reagent, which prevent the formation of disulfide bond between cysteine 208 at the dimer-dimer interface.

• Ion exchange is a useful and efficient method for purifying β-ureidopropionase mutations if IMAC and reverse IMAC are not sufficient to obtain a pure protein.

• Native-PAGE should be run for 3 hours at 150V so that the bands are well separated from each other.

• To get a good peak from light scattering, protein concentrations of 1-2mg/ml are optimal.

Further research can be done introducing other mutations of β-ureidopropionase, as such defined changes in important sites of β-ureidopropionase can give us a better understanding of the function of this enzyme and the mechanism of β-ureidopropionase. Finding a mutation that stabilizes β-ureidopropionase in a particular oligomeric state, may facilitate growth of larger, well-diffracting crystals of β-ureidopropionase and therefore the determination of the crystal structure of human β-ureidopropionase.

4. Appendix

4.1 List of abbreviations, formulas and symbols

(v/v) volume per volume

(w/v) weight per volume

APS Ammonium persulfate

C(Cys) Cysteine

D(Asp) Aspartic acid

DLS Dynamic light scattering

DNA Deoxyribonucleic acid

EDTA Ethylenediaminetetraacetic acid

HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

H(His) Histidine

IMAC Immobilized Metal Affinity Chromatography

IPTG Isopropyl β-D-1-thiogalactopyranoside

kDa Kilodalton

LB media Lysogeny broth media

Mw Molecular weight

Na-borate Boric acid buffer adjusted with sodium hydroxide

NTA Nitrilotriacetic acid

OD600nm Optical density at 600 nm

OPA Ortho-phthalaldehyde

PAGE Polyacrylamide gel electrophoresis

RNA Ribonucleic acid

S(Ser) Serine

SDS Sodium dodecyl sulfate

SLS Static light scattering

TB media Terrific broth media

TEMED N,N,N',N'-tetramethylethylenediamine

TEV Tobacco Etch Virus

Tris tris(hydroxymethyl)aminomethane

W(Trp) Tryptophan

4.2 Dynamic light scattering report

Avid-Nano\avidnano

Experiment Name: R130DS208R - Water - 20C Remark:

Sample Analysis Report

Date: 15/05/2017 15:52:26 Temperature: 20°C

Solvent: Water Solute: R130DS208R

Intensity: 362,214 counts/s Intercept: 0.773

Z - Av. Diameter: 12.51nm Standard Deviation: 15.46nm

Polydispersity: 123.54 % Polydispersity Index: 1.526

Peak No Mn. Diameter (nm)

Std. Dev. (nm) Polydisp. (%). Est. MW. (kDa) Intensity (%) Mass (%)

Avid-Nano\avidnano

Experiment Name: S208C - Water - 20C Remark:

Sample Analysis Report

Date: 10/05/2017 15:49:55 Temperature: 19.96°C

Solvent: Water Solute: S208C

Intensity: 302,739 counts/s Intercept: 0.787

Z - Av. Diameter: 39.45nm Standard Deviation: 48.73nm

Polydispersity: 123.52 % Polydispersity Index: 1.526

Peak No Mn. Diameter Std. Dev. (nm) Polydisp. (%). Est. MW. (kDa) Intensity (%) Mass (%) (nm)

1 10.15 5.82 57.36 64.64 34.63 99.96 2 349.96 377.26 107.80 263,036.40 54.65 0.04 3 8,887.23 5,035.69 56.66 Out of Range 10.72 0.00

4.3 Crystallization conditions

Table 6: Composition of additive mixtures

Mix name Composition

0.9M LiNaK 0.3 M Lithium sulfate, 0.3 M Sodium sulfate, 0.3 M

Potassium sulfate 0.02M Divalents II

0.005M Manganese(II) chloride tetrahydrate, 0.005M Cobalt(II) chloride hexahydrate , 0.005M Nickel(II) chloride hexahydrate, 0.005M Zinc acetate dihydrate

0.04M Alkalis 0.01M Rubidium chloride, 0.01M Strontium acetate,

0.01M Cesium acetate, 0.01M Barium acetate 0.02M Oxometalates

0.005M Sodium chromate tetrahydrate, 0.005M Sodium molybdate dehydrate, 0.005M Sodium tungstate dehydrate, 0.005M Sodium orthovanadate

0.02M Lanthanides

0.005M Yttrium(III) chloride hexahydrate, 0.005M Erbium(III) chloride hexahydrate, 0.005M

Terbium(III) chloride hexahydrate, 0.005M Ytterbium(III) chloride hexahydrate 1M Monosaccharides II

0.2M Xylitol, 0.2M Myo-Inositol, 0.2M

D-(-)-Fructose, 0.2M L-Rhamnose monohydrate, 0.2M D-Sorbitol

1M Amino acids II

0.2M DL-Arginine hydrochloride, 0.2M DL Threonine, 0.2M DL-Histidine monohydrochloride monohydrate, 0.2M DL-5-Hydroxylysine

hydrochloride, 0.2M trans-4-hydroxy-L-proline

0.4M Polyamines

0.1M Spermine tetrahydrochloride, 0.1M Spermidine trihydrochloride, 0.1M 1,4-Diaminobutane

dihydrochloride, 0.1M DL Ornithine monohydrochloride

Table 7: Composition of precipitant mixtures

Mix name Composition

Precipitant Mix 5 30% w/v PEG 3000, 40% v/v 1, 2, 4-Butanetriol, 2%

w/v NDSB 256

Precipitant Mix 6 25% w/v PEG 4000, 40% w/v 1,2,6-Hexanetriol

Precipitant Mix 7 20% w/v PEG 8000, 40% w/v 1,5-Pentanediol

Precipitant Mix 8 10% w/v PEG 20000, 50% w/v Trimethylpropane,

5. Acknowledgement

Thanks to Professor Doreen Dobritzsch from Uppsala University and Professor Zhen Xi from Nankai University for guiding and supervising my thesis project.

Thanks to Professor Xin Wen, Professor Baiquan Wang from Nankai University and Professor Mikael Widersten from Uppsala University for helping me make my study and life settled down in Uppsala.

Thanks to PhD student Dirk Maurer for introducing all the experiment processes to me and solving so many problems during the project.

6. Reference

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