University of Southern California Dissertations and Theses

Expression and purification of extracellular domain of human CD33 in Escherichia coli and Pichia pastoris

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Expression and purification of extracellular domain of human CD33 in Escherichia coli and Pichia pastoris

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Content Expression and Purification
of Extracellular Domain of Human CD33
in Escherichia coli and Pichia pastoris

by
Xuhang Dai

A Thesis Presented to the
FACULTY OF THE USC KECK SCHOOL OF MEDICINE
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfilment of
the Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR MEDICINE)

AUGUST 2021

Copyright 2021 Xuhang Dai
ii
Acknowledgements

I want to sincerely thank Dr. Ulmer for his patience, guidance and suggestion in the whole projects. This is
my first time to conduct biological experiments, so Dr. Ulmer’s patient instruction gave me chances to learn
necessary knowledge in this field.

I express my appreciation to Dr. Langen and Dr. Siemer, my thesis committee members. Dr. Langen’s
advice let me notice the importance of the deeper molecular mechanism of every step in the experimental
designs. Dr. Siemer’s suggestion make me develop better custom of experiment data saving and annotation.

I also want to express my gratitude to Alan Situ for his daily detailed instruction from molecular biology
experiments(PCR, Cell culturing) to protein purification(FPLC, HPLC).

I also would like to thanks my lab-mates Han Vu(Calibration of gel filtration column), Dai Zhai, and Jiaqi
Xiao for discussing and exchanging novel ideas of individual projects.

Since my personal ability and knowledge are limited, if you find some errors or want to discuss some details,
please feel free to contact me via email(xuhangda@usc.edu).

iii
Table of Contents
Acknowledgements ......................................................................................................................... ii
List of Tables .................................................................................................................................. v
List of Figures ................................................................................................................................ vi
Abstract ........................................................................................................................................ viii
Chapter 1 Introduction .................................................................................................................... 1
1.1 Alzheimer’s Disease ........................................................................................................................... 1
1.2 Microglia ............................................................................................................................................. 1
1.3 human CD33 ....................................................................................................................................... 1
1.4 Pichia pastoris expression system ....................................................................................................... 3
1.5 E.coli expression system ..................................................................................................................... 4
1.6 Tween-20 and Nonidet P-40 substitute ............................................................................................... 4
Chapter 2 Materials ......................................................................................................................... 6
2.1 Strains and Plasmids ........................................................................................................................... 6
2.2 Main reagents and enzymes ................................................................................................................ 6
2.3 Culture media ...................................................................................................................................... 6
2.4 Main machines .................................................................................................................................... 7
2.5 Primers ................................................................................................................................................ 7
Chapter 3. Cloning and expression of extracellular domain in Pichia pastoris .............................. 8
3.1 Introduction ......................................................................................................................................... 8
3.2 Methods............................................................................................................................................... 8
3.2.1 Primers design of expression vector of extracellular domain ...................................................................... 8
3.2.2 Construction of expression vector of extracellular domain ....................................................................... 11
3.2.3 Transformation of Pichia pastoris .............................................................................................................. 11
3.2.4 Protein Expression Screening .................................................................................................................... 12
3.2.5 Large scale expression of extracellular domain of CD33 .......................................................................... 12
3.3 Results ............................................................................................................................................... 14
3.3.1 Western blotting of pPIC9K-Msb2-CD33(D18-T232) ............................................................................. 14
3.3.2 Western blotting of pPIC9K-aM-CD33(D140-G266)-C169S ................................................................... 17
Chapter 4: Purification of extracellular domain of CD33 ............................................................. 19
4.1 Introduction ....................................................................................................................................... 19
4.2 Methods............................................................................................................................................. 19
4.2.1 IMAC column ............................................................................................................................................ 19
4.2.2 De-glycosylation and Dialysis ................................................................................................................... 20
4.2.3 HiTrap SP sepharose column ..................................................................................................................... 20
4.2.4 HiPrep™ 16/60 Sephacryl® S-100 HR column ........................................................................................ 21
4.2.5 Vivaspin® MWCO 5kDa(molecular weight cut-off) ................................................................................ 21
4.2.6 NMR sample preparation ........................................................................................................................... 21
4.3 Results ............................................................................................................................................... 22
iv
4.3.1 Chromatography of pPic9K-alphaMATING-KREAEA-Flag-CD33(D140-G266)-C169S-His6 ............. 22
4.3.2 SDS-PAGE of pPic9K-alphaMATING-KREAEA-Flag-CD33(D140-G266)-C169S-His6 ..................... 28
4.3.3 Chromatography of pPic9k-Msb2-CD33(D18-T232)-Flag-His6 .............................................................. 29
4.3.4 SDS-PAGE of pPic9k-Msb2-CD33(D18-T232)-Flag-His6 ...................................................................... 30
Chapter 5 Cloning and Expression of pET44-GB3R2-His6-TEV-CD33(D140-D298)-C169S ... 31
5.1 Introduction ....................................................................................................................................... 31
5.2 Methods............................................................................................................................................. 31
5.2.1 Primer design of expression vector of IgC2-TM domain .......................................................................... 31
5.2.2 Construction of expression vector of pET44-GB3R2-His6-TEV-CD33(D140-D298)-C169S ................. 31
5.2.3 Transformation tot BL21(DE3)-pLysS ...................................................................................................... 32
5.2.4 Expression of IgC2-TM domain ............................................................................................................... 32
Chapter 6 Purification of pET44-GB3R2-His6-TEV-CD33(D140-D298)-C169S ...................... 33
6.1 Methods............................................................................................................................................. 33
6.1.1 Lysis of cells .............................................................................................................................................. 33
6.1.2 IMAC column for getting His-tagged Protein of Interest .......................................................................... 33
6.1.3 Dialysis and TEV cleavage ........................................................................................................................ 34
6.1.4 IMAC column for obtaining cleaved Protein of Interest without His-tag ................................................. 34
6.1.5 High Performance Liquid Chromatography(HPLC) ................................................................................. 34
6.2 Results ............................................................................................................................................... 36
6.2.1 Chromatography of IMAC column ........................................................................................................... 36
6.2.2 SDS-PAGE of pET44-GB3R2-His6-TEV-CD33(D140-D298)-C169S .................................................... 38
6.2.3 HPLC of pET44-GB3R2-His6-TEV-CD33(D140-D298)-C169S ............................................................ 41
Chapter 7 Discussion .................................................................................................................... 42
7.1 Expression and Purification of IgV-IgC2 domain(Construct-1,2,3) ................................................. 42
7.2 Expression and Purification of IgC2-Linker domain(Construct-4, 5) .............................................. 44
7.3 Expression and Purification of IgC2-TM domain(Construct-6) ....................................................... 47
Chapter 8 Summary and Conclusion ............................................................................................ 49
References ..................................................................................................................................... 50

v
List of Tables

Table. 1 Calculation of the molecular weight of Peak-1 and Peak-2 obtained from Fig 12(a)………….…26
Table. 2 Calculation of the molecular weight of Peak-1 obtained from Fig 12(b)………………………..27

vi
List of Figures

Figure 1 pPIC9K and Endo Hf………………………………………………………………………………………………………..…3
Figure 2 pET44 gene map ………………………………………………………………………………………………….…………...4
Figure 3 Structural formula of Tween-20, from Sigma-Aldrich……………………………………………………………..5
Figure 4 Structural formula of Nonidet P-40 substitute, from Sigma-Aldrich…………………………………………….5
Figure 5 Western Blotting of Msb2-CD33(D18-T232)-Flag………………………………………………………………...14
Figure 6 Western blotting of pPIC9K-Msb2-CD33(D18-T232)-Flag-His6……………………………………………….15
Figure 7 Western blotting of pPIC9K-Msb2-CD33(D18-T232)-Flag-His10, with beta-ME……………………….…16
Figure 8 Western blotting of pPic9K-alphaMATING-KREAEA-Flag-CD33(D140-G266)-C169S-His6; Without
reducing agents; Provided by Alan Situ……………………………………………………………………………………….….17
Figure 9 Western blotting of pPic9K-alphaMATING-KREAEA-Flag-CD33(D140-G266)-C169S-Flag-
His10…………………………………………………….……………………………………………………………………………………….18
Figure 10 IMAC chromatography of pPic9K-alphaMATING-KREAEA-Flag-CD33(D140-G266)-C169S-
His6. ………………………………………….………………………………………………………………………………………………...22
Figure 11 SP HP chromatography of pPic9K-alphaMATING-KREAEA-Flag-CD33(D140-G266)-C169S-
His6. ………………………………………….………………………………………………………………………………………………...24
Figure 12 Gel filtration chromatography of pPic9K-alphaMATING-KREAEA-Flag-CD33(D140-G266)-C169S-
His6…………………………………………….………………………………………………………………………….…………………....26
Figure 13 SDS-PAGE of pPic9K-alphaMATING-KREAEA-Flag-CD33(D140-G266)-C169S-His6……………………28
Figure 14 IMAC chromatography of pPic9k-Msb2-CD33(D18-T232)-Flag-His6 withTween-20 in the
culturing media………………………………………………….…………………………………………………………………………..29
Figure 15 SDS-PAGE of pPic9k-Msb2-CD33(D18-T232)-Flag-His6………………………………………………………….…30
Figure 16 IMAC chromatography of pET44-GB3R2-His6-TEV-CD33(140-298)-C169S………………………….……36
vii
Figure 17 IMAC chromatography of cleaved pET44-GB3R2-His6-TEV-CD33(140-298)-C169S………………..…37
Figure 18 SDS-PAGE of pET44-GB3R2-His6-TEV-CD33(140-298)-C169S…………………………………………………..38
Figure 19 SDS-PAGE of TEV-cleaved pET44-GB3R2-His6-TEV-CD33(140-298)-C169S……………………………….39
Figure 20 SDS-PAGE of Pellet, Supernatant and Flowthrough(containing protein of interest) of pET44-
GB3R2-His6-TEV-CD33(140-298)-C169S…………………………………………………………………………………………………40
Figure 21 FPLC of the purified product of cleaved pET44-GB3R2-His6-TEV-CD33(D140-D298)-C169S………41

viii
Abstract

Alzheimer’s Disease (AD) is affecting millions of people worldwide. Innate immune response and
inflammation mediated by microglia and astrocytes may play a role in AD according to recent studies.
CD33 is expressed on the membrane of microglia in the brain, composed of extracellular domain(IgV and
IgC2), a single-pass transmembrane domain(TM) and a cytosolic domain(CS). Genome-wide association
studies(GWAS) show that CD33 is one of the genes associated with AD.

However, little is known about the real structure and function of CD33 in microglia up to now. In this study,
extracellular domain of human CD33 was both expressed in E.coli and Pichia pastoris. The aim is to express
isotope-labeled IgV-IgC2, IgC2-linker and IgC2-TM domain for further solution-state NMR analysis.

To date, 15-N labeled IgC2-Linker recombinant protein was successfully expressed and purified. The
optimization of culturing condition and addition of detergent P-40(nonidet P-40 substitute) can improve the
expression level of IgC2-Linker in Pichia pastoris as well as purification process. The IgC2-TM
recombinant protein was also expressed and purified in E.coli. The expression and purification results for
IgV-IgC2 domain was not satisfying. The reason could be the IgV domain’s binding affinity to
polysaccharide, glycoprotein and glycolipid. It is promising to produce isotope-labeled IgC2-Linker and
IgC2-TM with these improvements and constructs.

1
Chapter 1 Introduction
1.1 Alzheimer’s Disease
Alzheimer’s Disease(AD), a type of degenerative brain disease, the main cause of dementia, accounting for
an estimated 60% to 80% of actual cases, was affecting 5.8 million American people
1
in 2020, and around
50 million people worldwide
2
.
There are two main neuropathological characterizations of Alzheimer’s Disease. One is the extracellular
formation of amyloid plaques made up of Amyloid-beta peptides; the other is the intracellular amassing of
neurofibrillary tangles, basically aggregations of hyperphosphorylated tau protein
3
.
Animal models and clinical case studies robustly indicate the significant role of inflammation in the
pathogenesis in AD
4
. The binding of pattern recognition receptors on microglia and astroglia with
aggregate proteins(Amyloid-beta, etc.) will trigger an innate immune response which might also contribute
to AD
5
.

1.2 Microglia
Microglia are known as the resident innate immune cells of the central nervous system, and their function
is always believed to be normally protective in the brain as phagocytes maintaining tissue homeostasis and
clearing up amyloid plaques. However, activated microglia can be harmful, injuring neurons directly or
indirectly
6
. This Two-edged sword model is reviewed well by D. Hansen, et al
7
.

1.3 human CD33
Myeloid cell surface antigen CD33, belonging to Sialic-acid-binding immunoglobulin-like lectin (Siglec)
family, is expressed on microglia in the brain. Alpha-2,3- linked sialic acid-bearing glycans are
preferentially bound to CD33, while alpha-2,6-linked sialic acid-bearing glycans will be bound more avidly
8
.
2
According to GWAS studies, CD33 is associated with Alzheimer’s Disease
9,10
. Different SNPs may
increase, decrease or non-related to Alzheimer’s Disease risk. For example, the polymorphism (rs12459419)
9
causing loss of Exon2 which encodes binding domain(IgV) may decrease the risk. However, little is
known about the acutal role of CD33 in microglia.
The longest transcript is studied in this project (NM_001772.4). The amino acid sequence(364aa in total )
encoded by each exon is shown as blew.
Exon-1: aa 1-12
MPLLLLLPLLWA
Exon-2: aa 13-139
GALAMDPNFWLQVQESVTVQEGLCVLVPCTFFHPIPYYDKNSPVHGYWFREGAIISRDSPVATN
KLDQEVQEETQGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFRMERGSTKYSYKSPQLSVHVT
Exon-3: aa 140-232
DLTHRPKILIPGTLEPGHSKNLTCSVSWACEQGTPPIFSWLSAAPTSLGPRTTHSSVLIITPRPQDHG
TNLTCQVKFAGAGVTTERTIQLNVT
Exon-4: aa 233-248
YVPQNPTTGIFPGDGS
Exon-5: aa 249-280
GKQETRAGVVHGAIGGAGVTALLALCLCLIFF
Exon-6: aa 281-308
IVKTHRRKAARTAVGRNDTHPTTGSASP
Exon-7: aa 309-364
KHQKKSKLHGPTETSSCSGAAPTVEMDEELHYASLNFHGMNPSKDTSTEYSEVRTQ

3
1.4 Pichia pastoris expression system
Pichia pastoris is a well-established heterologous protein expression system. Compared with E.coli,
disulfide formation and glycosylation is available in Pichia pastoris.
pPIC9K plasmid is used in this study. The picture is from Invitrogen. 5’AOX1 gene plays the main role in
utilizing Methanol during growth. HIS4 gene is used for selection when competent Pichia pastoris cells are
his4-∆, as the one we used in this study. Ampicillin resistance gene is usually used in selection in E.coli
harboring this plasmids in early-stage experiments. Kanamycin(Geneticin) resistance gene can be used for
selection of multicopy because the level of Geneticin resistance is roughly correlated with the number of
Kanamycin resistance genes inserted into the genome. Linearized plasmids can be integrated into the
genome of Pichia pastoris via homologous recombination. In this thesis, PmeI is used to linearize pPIC9K
plasmid in the middle of HIS4 gene sequence.

Figure 1 pPIC9K and Endo Hf. (a) pPIC9K gene map; (b) Function of Endo Hf

Endo Hf, a recombinant protein fusion of Endoglycosidase H and maltose binding protein(MBP), is used
in experiments in this thesis to de-glycosylate secreted protein. The cleavage happens within the chitobiose
core of high mannose and hybrid oligosaccharides from N-linked glycosylation.
A signal peptide is a short peptide usually presenting at the N-terminus to guide the protein toward the
secretory pathway. Alpha mating factor secretion signal sequence(aM) exists in pPIC9K for wide
application. In this study, Msb2
11
, a native signal peptide in Pichia pastoris is also tried in order to express
IgV-IgC2 domain because of previous failure expression of it with aM sequence.
(a)
(b)
4
1.5 E.coli expression system
E.coli is widely used as one of the most common expression system for fast, large-scale and cost-friendly
expression. Both soluble and insoluble proteins can be expressed in E.coli.
pET44 plasmid was used in this study. The figure is from Novagen. Isopropyl β-d-1-
thiogalactopyranoside(IPTG) was used to induce the expression of recombinant protein.

Figure 2 pET44 gene map
Tobacco etch virus protease(TEV) is used in this study to cut off the tag from protein of interest by cleaving
Glu-Asn-Leu-Tyr-Phe-Gln-(Gly/Ser) sequence constructed in the recombinant protein.

1.6 Tween-20 and Nonidet P-40 substitute
Both Tween-20 and Nonidet P-40 substitute(P-40) are nonionic and non-denaturing detergent applied in
biochemistry for purification and other application.

In this study, they are both added to the culture media to improve protein expression
12,13
. Their structures
are shown in Figure 3and Figure 4.(from company websites.)

5

Figure 3 Structural formula of Tween-20, from Sigma-Aldrich

Figure 4 Structural formula of Nonidet P-40 substitute, from Sigma-Aldrich

6
Chapter 2 Materials
2.1 Strains and Plasmids
(1) Pichia pastoris strain: BG12 Histidine auxotrophic Pichia (Komagataella phaffii) expression
strain; Genotype: his4-∆1
(2) E.coli strain-1: XL10-Gold® ultracompetent cells, high efficiency of transformation
(3) E.coli strain-2: BL21(DE3)pLysS Competent Cells; high efficiency of protein expression
(4) Plasmid for Pichia pastoris: pPIC9K
(4) Plasmid for E.coli: pET44

2.2 Main reagents and enzymes
Tween-20: AMRESCO; Nonidet
®
P-40 Substitute: VWR Life Science
BamHI, EcoRI, XhoI, Endo Hf, TEV, Q5 High-Fidelity 2X Master Mix: New England BioLabs
Anti-Flag(DYKDDDDK Tag) antibody, CDP-Star, Emerald-II™ Enhancer: Thermo Fisher
Blotting-Grade Blocker: BIO-RAD

2.3 Culture media
(1) BMG (Buffered Minimal Glycerol) and BMM (Buffered Minimal Methanol) media for 1 Liter
-100 mL of 1 M potassium phosphate buffer, pH 6.0 (-> final concentration 100 mM)
-100 mL 10X (Yeast Nitrogen Base without Ammonium Sulfate)YNB without amino acids and
with ammonium sulfate (-> final concentration 2.5 g/L)
-2 mL 500X biotin (-> final concentration 4·10
-5
%)
-50 mL 20X methanol (-> final concentration 0.5%) or 20X glycerol (-> final concentration 1%)
7
-All of these buffer stocks are filtered and sterile.
(2) LB culture: LB broth 20g/L
(3) Minimal Media for 1 Liter
50mL 20X MM stock(120g/L Na2HPO4 7H2O; 60g/L KH2PO4; 10g NaCl; 10g NaCl); 0.5g NH4Cl;
930mL DI water; 1mL 1M MgSO4; 1mL 0.1 M CaCl2; 500uL Thiamine HCl(20mg/mL); 20 mL
20% Glucose; 500uL Ampicillin(1g/10mL); 1mL Chloramphenicol(34mg/mL).

2.4 Main machines
FPLC: ÄKTAprime plus
HPLC: Shimadzu UFLC
2.5 Primers
Primers were synthesized by company Integrated DNA Technologies(IDT).

8
Chapter 3. Cloning and expression of extracellular domain in Pichia
pastoris

3.1 Introduction
In Chapter 3, three constructs of IgV-IgC2 domain, and two constructs of IgC2-Linker domain
were designed. Each was expressed in Pichia pastoris to produce secreted extracellular domain of
human CD33.

3.2 Methods

3.2.1 Primers design of expression vector of extracellular domain

Primers were designed according to the human CD33 gene sequence(NM_001772.4) provided on
NCBI website.

3.2.1.1 IgV-IgC2(VC2) domain
IgV-IgC2(VC2) is the region ranging from 40bp to 736bp, contained in Exon2 and Exon3,
encoding amino acids from 18D to 232T. The details and primers are shown respectively as blew.

(1)Construct -1: pPIC9K-Msb2-CD33(D18-T232)-Flag
(BamHI&EcorI)
Forward primer: [fwd-Msb2-CD33-D18]
9
5’-GAT ATA GGA TCC ATG ATT AAT TTA AAC TCC TTT CTT ATA CTT ACA GTA ACA
CTG TTA TCT CCA GCT TTG GCA gat cca aat ttc tgg ctg c-3’
Backward primer(Reverse complement): [bwd-CD33-T232-Flag]
5’-AAGGGCGAATTCTTACTTGTCGTCATCGTCTTTGTAGTCTGAACCggtgacgttgagctggat
-3’

(2) Construct-2: pPIC9K-Msb2-CD33(D18-T232)-Flag-His6
(BamHI&EcorI)
Forward primer:
the same as [fwd-Msb2-CD33-D18]
Backward primer(Reverse complement): [bwd-CD33-T232-Flag-His6]
5‘AAGGGCCGCGGCCGCTTAAGAGTGGTGATGATGGTGATGTCCACCCTTATCATCGT
CATCTTTGTAATCGGAATTCCCggtgacgttgagctggat-3’

(3) Construct-3: pPIC9K-Msb2-CD33(D18-T232)-TEV-Flag-His10
(BamHI&EcorI)
Forward primer:
the same as [fwd-Msb2-CD33-D18]
Backward primer(Reverse complement):
the same as [bwd-aM-IgC2-His10]

10
3.2.1.2 IgC2-Linker domain(C169S mutation)
IgC2-Linker domain(C2L) is the region ranging from 457bp to 912bp, contained in Exon2,Exon3,
Exon4 and Exon5, encoding amino acid from D140 to G266. Only one single bp is included in
Exon2.

(1) Construct-4: pPIC9K-alphaMATING-KREAEA-Flag-CD33(D140-G266)-C169S-His6
This construct was provided by Technician Alan Situ.

(2)Construct-5:pPIC9K- alphaMATING-KREAEA-Flag-CD33(D140-G266)-C169S-TEV-Flag-
His10
(seq march31) (BamHI&EcorI)

Forward primer: [fwd-aM-CD33-D18]
5’-CGC GG ATC C AAACG ATG AGA TTT c-3’
Backward primer(Reverse complement): [bwd-CD33-G266]
5’- CCGGAATTCccaccagctcctccaatggc-3’

11
3.2.2 Construction of expression vector of extracellular domain
Primers were used with the template of CD33 original sequence(BamHI site removed) in the PCR.
The PCR products and plasmid pPIC9K were digested with restriction enzymes BamHI and EcoRI.
In addition, antarctic phosphatase was utilized to The digested fragments were purified by agarose
gel(1.5% or 1.0%). After being purified, both the inserts and vectors were ligated Ligase overnight.
The ligated recombinant expression plasmids were then transformed into competent E.coli
cells(XL10). The colonies grew on the LB Lenox Agar plate containing Ampicillin(0.1%v/v).
Positive colonies were picked to grow overnight in order to extract plasmids for DNA sequencing.
The extracted DNA was sent to GENEWIZ company for sequencing after verification via double
enzyme digestion. The plasmids of expected insert sequences were transformed into E.coli cells
(XL10). Plasmids of expression vector required for transformation of P.pastoris were obtained by
100mL LB culture, and the concentrations were measured by UV spectrometer.

3.2.3 Transformation of Pichia pastoris
The constructed plasmids were linearized by restriction enzyme PmeI. Purified linearized plasmids
were subsequently transformed in to competent cells of Pichia pastoris(BG12 His4-) via
electroporation, according to the standard protocol from Invitrogen Company. After
electroporation, 1 mL Sorbitol was added to the competent cells. Then, the tubes of transformed
Pichia pastoris were shaken for 1 hour at 30 °C. Subsequently, these transformants grew on the
YPD agar plates. The competent cells, BG12 His 4-, cannot synthesize histidine themselves, so
they cannot grow on the YPD plates lacking histidine. The HIS4 gene(histidine dehydrogenase) is
included in pPIC9K plasmid, and can be integrated into the genome of Pichia pastoris by
homologous recombination. Only His + transformants are able to grow on the YPD plates.
12
3.2.4 Protein Expression Screening
First, nine His+ transformant colonies picked from the YPD plates were grown in 4mL BMGY
overnight. The next morning, their OD(2-6) was measured respectively. Different volumes, in
order to achieve OD600=1.0 in BMMY culture, of Pichia BMGY culture were spined down, and
the supernatant was removed. The pellets were resuspended with 4mL BMMY, and grew for 2
days at 30°C. 20uL 100% Methanol was added to make final 0.5% Methanol(v/v). Also, 30%
glycerol stocks were made from BMGY samples.

After two days, the supernatant was collected for Western Blotting. Briefly, 15uL supernatant
added with 2ul Beta-mercaptoethanol and 5uL 4X SDS loading buffer was denatured at 70°C for
15min. After SDS-PAGE and membrane transfer, the nitrocellulose membrane was blocked with
5% milk/TBST for 1 hour. After washing three times with TBST, 10mL anti-Flag antibody (1:1000
v/v, 30%BSA, TBST) was used to incubate the membrane. Finally, after washing 3 times with
TBST, CDP-Star® Substrate with Emerald-II™ Enhancer(3mL:150uL) were utilized to visualize
the bands.
The strains with bands of the correct size and most brightness were selected for the following
large-scale expression.

3.2.5 Large scale expression of extracellular domain of CD33
For the starting culture, inoculation of 4mL BMG from glycerol stocks was done at 30°C for 24
hours in a shaking incubator(200rpm).

13
On the second day, the starting culture was spined down at 2,500xg for 5 mins, and the supernatant
was discarded. The pellet was resuspended with fresh BMG medium. The resuspended BMG
medium was used to inoculate 500mL BMG media in a baffled 2-liter flask. The culture was grown
overnight at 30°C in a shaking incubator at 200rpm.

On the third day, the OD600 of the culture was measured, which should be around 5-7. Enough
BMMY culture was spined down to achieve final OD600=1.0 in 1Liter BMMY culture medium.
The supernatant was discarded, and the pellet was resuspended with 1 liter fresh BMMY culture
in each 4-liter flask.

The BMMY culture was grown at 27°C for 48 hours in a shaking incubator(200rpm). 100%
Methanol was replenished to each 1-liter culture every 12 hours to reach a final concentration of
0.5%.

On the fifth day, the Pichia pastoris supernatant was harvested by centrifugation at 4°C for
20min(4,000g). The recombinant extracellular domain of CD33 was secreted into the supernatant.

14
3.3 Results
3.3.1 Western blotting of pPIC9K-Msb2-CD33(D18-T232)
3.3.1.1 pPIC9K-Msb2-CD33(D18-T232)-Flag

Figure 5 Western Blotting of Msb2-CD33(D18-T232)-Flag
Lane 1: Ladder;
Lane2-10: No.1 to No.9 samples;
Anti-Flag antibody(1:1000, 30%BSA, TBST buffer)
Bands around 42kDa can be observed.

24kDa
72kDa
42kDa
15
3.3.1.2 Western blotting of pPIC9K-Msb2-CD33(D18-T232)-Flag-His6

Figure 6 Western blotting of pPIC9K-Msb2-CD33(D18-T232)-Flag-His6

Figure 6(a): original screening of 9 samples, without addition of DTT or beta-ME.
Dithiothreitol (DTT) and 2-Mercaptoethanol(beta-ME) are reducing agents to break disulfides.
Lane-1: Ladder; Lane 2-10: No.1-9 samples.
Smear bands and faint bands at 42kDa can be observed.
Figure 6 (b): No. 2, 6, 7 and 8 samples as shown in Figure 6(a) were selected.
Lane-1: Ladder; Lane2-5: No. 2, 6, 7, 8 samples without DTT or beta-ME;
Lane 6: Blank; Lane 7-10: No. 2, 6, 7, 8 samples with DTT.
For the samples without DTT/beta-ME, only smear samples can be observed. For the samples with DTT, bands around 42kDa were
shown.

(a) Western blotting of
pPIC9K-Msb2-CD33(D18-T232)-Flag-His6; without DTT or
beta-ME

(b) Western blotting of
pPIC9K-Msb2-CD33(D18-T232)-Flag-His6; No.2,6,7,8 were
selected; both with DTT

24kDa
72kDa
42kDa
42kDa
24kDa
72kDa
16
3.3.1.3 Western blotting of pPIC9K-Msb2-CD33(D18-T232)-Flag-His10

Figure 7 Western blotting of pPIC9K-Msb2-CD33(D18-T232)-Flag-His10, with beta-ME.
Lane-1: Ladder;Lane-2: Positive control; Lane3-10: No.1-8 samples.
The band of positive control was very bright. Faint bands at 42kDa were also shown on the membrane.

24kDa
72kDa
42kDa
17
3.3.2 Western blotting of pPIC9K-aM-CD33(D140-G266)-C169S
3.3.2.1 pPic9K-alphaMATING-KREAEA-Flag-CD33(D140-G266)-C169S-His6

Figure 8 Western blotting of pPic9K-alphaMATING-KREAEA-Flag-CD33(D140-G266)-C169S-His6; Without reducing agents;
Provided by Alan Situ.
Lane-1: Ladder; Lane-2-10: No.1-9 samples; Antibody: Anti-Flag tag(1:1000 v/v, 30%BSA,TBST)
Smear bands and bands at 18kDa were observed in all lanes. Bands at 31kDa were shown in No. 4, 6, 7.

24kDa
72kDa
31kDa
18kDa
18
3.3.2.2 pPic9K-alphaMATING-KREAEA-Flag-CD33(D140-G266)-C169S-Flag-His10

Figure 9 Western blotting of pPic9K-alphaMATING-KREAEA-Flag-CD33(D140-G266)-C169S-Flag-His10Lane-1: Ladder
Lane-2: Control but no signals
Lane-3-10: No.1-8 samples
The positive control band was not observed. Bands at 31kDa could be seen in other lanes except Lane3(No.2 sample)

24kDa
72kDa
31kDa
19
Chapter 4: Purification of extracellular domain of CD33
4.1 Introduction
All constructs were expressed, and then their supernatant was purified by IMAC column at first to obtain
polyhistidine-tagged protein. After confirmation with SDS-PAGE staining, polyhistidine-tagged proteins
of correct molecular weight were then deglycosylated and purified subsequently with SP column(cation
exchange column) and Gel filtration column(size exclusion column).
4.2 Methods
4.2.1 IMAC column
The aim of this step is to obtain polyhisitdine-tagged proteins.
The AΚTA® purifier Prime plus Fast Performance Liquid Chromatography(Amersham Biosciences,
Freiburg), also known as FPLC, was utilized in the whole process of protein purification.

HiTrap IMAC(Immobilized Metal Chelate Chromatography) HP column (cat no. 17-0409-03) column was
used to purify the supernatant collected from Pichia Pastoris culture. IMAC column, also named Nickel
column, can purify protein with hexahistidine tag via nickel-histidine reaction regardless of secondary
structure.

The purification process started after doing ‘system wash’ by Milli-Q water. Then attaching the IMAC
column(5mL), washing out 20% Ethanol with Milli-Q water, and charging it with 5mL 0.1M NiSO4 at
2mL/min were done.

Next, IMAC column equilibrate with wash buffer 1: 50 mM KH2PO4/K2HPO4, pH 8.0, 300 mM KCl, 20
mM imidazole. After equilibration, the supernatant could be loaded onto the column at 1-2mL/min at cold
room.
20

When the loading finished, the column was washed with 5CV with wash buffer 1 until a stable baseline
was shown. Then the column was washed by buffer 2:50 mM KH2PO4/K2HPO4, pH 8.0, 300 mM KCl,
40 mM imidazole to remove weakly bound proteins.

Finally, Elution buffer: 50 mM KH2PO4/K2HPO4, pH 8.0, 300 mM KCl, 300 mM imidazole was used to
elute well bound protein.

All the peaks were collected separately during each washing step for the following SDS-PAGE analysis.

After the purification process finished, Milli-Q water was used to wash other things out, and equilibrate
with 20% ethanol for long storage.

4.2.2 De-glycosylation and Dialysis
The aim is to remove the glycosylation and check the molecular weight of nonglycosylated protein.
50 uL NEB EndoHf (1,000,000 units/ml) was added to the eluate.
Then the eluate with EndoHf was dialyzed overnight against 4 Liters of Buffer A: 25 mM citric acid/sodium
citrate, pH 5.0, at room temperature.

4.2.3 HiTrap SP sepharose column
SP column is a cation exchange column. The aim is to separate proteins by their net positive charges.
SP Sepharose column is based on the strong cation exchange principle.
Since our sample was relatively clean, we could use SP column for this step; otherwise, impurities would
block the column’s filter with 34 μm particle size.
Before the purification process, all prework was the same as [4.2.1].
21
First, the SP column was equilibrated with Buffer A: 25 mM citric acid/sodium citrate, pH 5.0 at 5 min/mL.
Then, the mixture of protein and EndoH was loaded onto the SP column at 2mL/min.
After loading, the column was washed with 6-8V buffer A.
Subsequently, elution was done with a linear gradient to Buffer B: 25 mM citric acid/sodium citrate, pH
5.0, 500 mM NaCl in 8 CV. Every single peak was collected.
Finally, the column was washed with 25 mM citric acid/sodium citrate, pH 5.0, 1 M NaCl, and store in H2O.

4.2.4 HiPrep™ 16/60 Sephacryl® S-100 HR column
The purpose is to separate the peaks obtained from SP column by their size, and then calculate their
molecular weight.
First, the HiPrep™ 16/60 Sephacryl® S-100 HR column was equilibrated overnight with buffer: 50mM
sodium phosphate monobasic/sodium phosphate dibasic buffer, pH=7.4, 150mM NaCl.
The protein collected from SP column was injected into the machine at “loading” position. Then, the
machine was kept “injection” position during the whole purification process.
All the peaks were collected.

4.2.5 Vivaspin® MWCO 5kDa(molecular weight cut-off)
Only molecules smaller than 5kDa could flow through the membrane. The aim is to exchange buffer and
concentrate protein solution to around 350uL.
Each peak was concentrated and exchanged with buffer: 50mM sodium phosphate monobasic/sodium
phosphate dibasic buffer(pH=7.4) using Vivaspin® MWCO 5kDa for 4-5times. The final volume was
300uL, and the concentration was measured by UV-spectrometer.

4.2.6 NMR sample preparation
Correspondent D2O was added according to the accurate volume of the protein samples. The mixture was
then transferred into an NMR tube, labeled, and kept in the 4 °C fridge.
22
4.3 Results
4.3.1 Chromatography of pPic9K-alphaMATING-KREAEA-Flag-CD33(D140-G266)-C169S-His6
4.3.1.1 IMAC purification

Figure 10 IMAC chromatography of pPic9K-alphaMATING-KREAEA-Flag-CD33(D140-G266)-C169S-His6
(a) Without EndoH in culturing media
No EndoH was added in the culture medium. Cells grew at 27°C for 2 days(0.5% Methanol induction).
Peak-1 and Peak-2 were collected from Wash Buffer I: 50 mM KH2PO4/K2HPO4, pH 8.0, 300 mM KCl, 20 mM imidazole.
Proteins which were not well bound to the IMAC column were washed out in Wash Buffer I. No peak was observed in Wash
Buffer II: 50 mM KH2PO4/K2HPO4, pH 8.0, 300 mM KCl, 40 mM imidazole.

Peak-1
Peak-2
Peak-Elution
23

(b) With 100ul EndoH in the culture media for last four hours
Peak-1 and Peak-2 were collected from Wash Buffer I: 50 mM KH2PO4/K2HPO4, pH 8.0, 300 mM KCl, 20 mM imidazole. No
peak was observed in Wash Buffer II: 50 mM KH2PO4/K2HPO4, pH 8.0, 300 mM KCl, 40 mM imidazole.

Peak-1
Peak-2
Peak-Elution
24
4.3.1.2 SP HP column

Figure 11 SP HP chromatography of pPic9K-alphaMATING-KREAEA-Flag-CD33(D140-G266)-C169S-His6
(a) SP HP column; cleaved by Endo H during dialysis.
Elution peak from IMAC column was cleaved by Endo H during dialysis (pH=5.0) at room temperature.
Gradient program(40mL), combination of Buffer A and Buffer B, was applied. Buffer A: 25 mM citric acid/sodium citrate, pH 5.0;
Buffer B: 25 mM citric acid/sodium citrate, pH 5.0, 500 mM NaCl.
The elution peak came in 100% Buffer B.

Peak-1
25

(b) SP HP column; cleaved by Endo H in the culturing media
In this situation, EndoH was added in the culture media, so the Elution peak from IMAC was directly dialyzed in Buffer A 25 mM
citric acid/sodium citrate( pH 5.0) at room temperature.
A double peak was observed at 100% Buffer B. Peak-1 and Peak-2 were collected separately.

Peak-1
Peak-2
26
4.3.1.3 HiPrep™ 16/60 Sephacryl® S-100 HR column

Figure 12 Gel filtration chromatography of pPic9K-alphaMATING-KREAEA-Flag-CD33(D140-G266)-C169S-His6
(a) Gel filtration chromatography, cleaved by Endo H during dialysis
In this situation, Protein was cleaved during dialysis, and purified by SP column.
The filtered eluate from HP column was applied for gel filtration. The elution volume(Ve) of Peak-1 was 38mL; The elution
volume(Ve) of Peak-2 was 53mL.

Table. 1 Calculation of the molecular weight of Peak-1 and Peak-2 obtained from Fig 12(a).
The monomer molecular weight including Flag-tag(DYKDDDDKSG) and Hexahistidine-tag(SHHHHHH).
The actual Ve of Peak 2 is 53 mL; The predicted Ve of dimer is 54.83 mL.

Protein MW Log(MW) Ve Vo Vc Vavg = (Ve-Vo)/(Vc-Vo)
Aprotinin 6500 3.812913357 96.04 36.07 120 0.714524008
Ribonuclease A 13700 4.136720567 70.23 36.07 120 0.407005838
Carbonic Anhydrase 29000 4.462397998 58.49 36.07 120 0.267127368
Ovalbumin 44000 4.643452676 50.02 36.07 120 0.166209937
Conalbumin 75000 4.875061263 44.82 36.07 120 0.104253545
IgC2 (D140-T266) C169S 15269.91 Log(MW) Actual Ve Vo Vc Actual Vavg Predicted Ve Calc Vavg Power MW Power
monomer 15269.91 4.183836477 36.07 120 68.15681418 0.38230447
dimer 30539.82 4.484866473 36.07 120 54.83201209 0.223543573
Peak 1 38 36.07 120 0.022995353 576394.1802
Peak 2 53 36.07 120 0.201715715 34874.32894
Peak-1
Peak-2
27

Figure 12 (b) Gel filtration chromatography, cleaved by Endo H in the culturing media
In this situation, the protein were cleaved during the last 4 hours in culture medium.
The elution volume of Peak-1 is 55 mL.

Table. 2 Calculation of the molecular weight of Peak-1 obtained from Fig 12(b).
The monomer molecular weight including Flag-tag(DYKDDDDKSG) and Hexahistidine-tag(SHHHHHH).
The actual Ve of Peak 1 is 56 mL; The predicted Ve of dimer is 54.83 mL.

Protein MW Log(MW) Ve Vo Vc Vavg = (Ve-Vo)/(Vc-Vo)
Aprotinin 6500 3.812913357 96.04 36.07 120 0.714524008
Ribonuclease A 13700 4.136720567 70.23 36.07 120 0.407005838
Carbonic Anhydrase 29000 4.462397998 58.49 36.07 120 0.267127368
Ovalbumin 44000 4.643452676 50.02 36.07 120 0.166209937
Conalbumin 75000 4.875061263 44.82 36.07 120 0.104253545
IgC2 (D140-T266) C169S 15269.91 Log(MW) Actual Ve Vo Vc Actual Vavg Predicted Ve Calc Vavg Power MW Power
monomer 15269.91 4.183836477 36.07 120 68.15681418 0.38230447
dimer 30539.82 4.484866473 36.07 120 54.83201209 0.223543573
Peak 1 56 36.07 120 0.237459788 28247.99189
Peak-1
28
4.3.2 SDS-PAGE of pPic9K-alphaMATING-KREAEA-Flag-CD33(D140-G266)-C169S-His6

Figure 13 SDS-PAGE of pPic9K-alphaMATING-KREAEA-Flag-CD33(D140-G266)-C169S-His6
1: Ladder
2: IMAC; Peak1; Wash Buffer I; without EndoH in media; Pre-P40 in media
3: IMAC; Peak2; Wash Buffer I; without EndoH in media; Pre-P40 in media
4: IMAC; Elution buffer; without EndoH in media; Pre-P40 in media
5: IMAC; Peak1; Wash Buffer I; with EndoH in media; Pre-P40 in media
6: IMAC; Peak2; Wash Buffer I; with EndoH in media; Pre-P40 in media
7: IMAC; Elution buffer; with EndoH in media; Pre-P40 in media
8: Blank
9: SP column; Peak-1; 100%B; with EndoH in media; Pre-P40 in media
10: SP column; Peak-2; 100%B; After Peak-1; with EndoH in media; Pre-P40 in media
11: SP column; Only Peak; 99%B; after dialysis with EndoH 15uL overnight at room temperature; without EndoH in media; Pre-
P40 in media
12: Nothing

1 2 3 4 5 6 7 8 9 10 11 12

24kDa
72kDa
15kDa
30kDa
29
4.3.3 Chromatography of pPic9k-Msb2-CD33(D18-T232)-Flag-His6

Figure 14 IMAC chromatography of pPic9k-Msb2-CD33(D18-T232)-Flag-His6 withTween-20 in the culturing media.
In this situation, the tween-20(0.2g/L) was added to the culture medium.
Only one peak was observed in Wash Buffer I: 50 mM KH2PO4/K2HPO4, pH 8.0, 300 mM KCl, 20 mM imidazole.

Peak-1
30
4.3.4 SDS-PAGE of pPic9k-Msb2-CD33(D18-T232)-Flag-His6

Figure 15 SDS-PAGE of pPic9k-Msb2-CD33(D18-T232)-Flag-His6
1: Ladder
2: Blank
3: CD33(140-266), pre P-40; Wash buffer I; with betaME
4. CD33(140-266), pre P-40; Elution buffer; with betaME
5. Pet44-CD33(140-298); Elution buffer; with betaME
6. Msb2-CD33(18-232); Tween-20; Wash I buffer: with betaME
7: Blank
8: CD33(140-266), pre P-40; Wash buffer I
9: CD33(140-266), pre P-40; Elution buffer
10: Pet44-CD33(140-298); Elution buffer
11: Msb2-CD33(18-232); Tween-20; Wash I buffer
12: Blank

1 2 3 4 5 6 7 8 9 10 11
12
24kDa
72kDa
Msb2-CD33(18-232); Tween-20;
Wash I buffer; without beta-ME

Lane-6: Msb2-CD33(18-232); Tween-20;
Wash I buffer; with beta-ME

31
Chapter 5 Cloning and Expression of pET44-GB3R2-His6-TEV-
CD33(D140-D298)-C169S
5.1 Introduction
In Chapter 5, the expression vector of IgC2-TM domain was designed and expressed in E.coli. The
aim was to produce enough recombinant protein with both IgC2 and transmembrane(TM) domain
to prepare NMR samples.

5.2 Methods
5.2.1 Primer design of expression vector of IgC2-TM domain
IgC2-TM domain is the region ranging from 457bp to 933bp, contained in Exon3, 4, 5 and 6,
encoding amino acids from D140 to D298.
Construct-6: pET44-GB3R2-His6-TEV-CD33(D140-D298)-C169S
Forward primer: [fwd-BamHI-TEV-C2TM-D140]:
5’-CGCGGA TCC GAA AAC CTG TAT TTC CAG ggc gac ttg acc cac agg ccc-3’
Backward primer(reverse complement): [bwd-D298-XhoI]
5’-CCGCTCGAGTTAgtcattcctgcccactgc-3’

5.2.2 Construction of expression vector of pET44-GB3R2-His6-TEV-CD33(D140-D298)-C169S
The CD33 original sequence(BamHI removed, C169S), done by Technician Alan Situ, was used
as the template in the PCR. The recombinant pET44 harboring GB3R2 sequence was from Alan
Situ. Both the PCR product and pET44 templates were digested with restriction enzymes BamHI
and XhoI. Other steps were similar to [3.3.2] in Chapter 3.
32
5.2.3 Transformation tot BL21(DE3)-pLysS
The plasmids with the correct sequence were transformed into CaCl2 competent cells BL21(DE3)-
pLysS. The transformed cells grew on the LB agar plate with both Ampicillin and
Chloramphenicol. The positive transformant colonies were ready for inoculation.

5.2.4 Expression of IgC2-TM domain
For the starting culture, 2mL LB Lennox culture was inoculated with a single positive transformant
colony, with 100ug/mL Ampicillin and 34ug/mL Chloramphenicol. The starting culture grew at
37 °C in the incubator for around 6-8 hours.
Then, for each 1 Liter expression culture, 25mL starter minimal medium(MM) culture was
inoculated with 0.25mL of LB starting culture(1% v/v), with 50ug/mL Ampicillin and 34ug/mL
Chloramphenicol. The starter MM culture grew overnight at 37 °C for 4 hours.
On the second day, each 25mL starter MM culture was spun down and resuspended with fresh
MM medium for 1Liter MM culture(2.5% v/v).
On the OD600 of 0.85, IPTG, at a final concentration of 0.5mM, was used to induce protein
expression. The 1 Liter MM culture grew at 37C incubator with shaking at 200rpm.
The protein of interest (IgC-TM) was inside cells. The culture was spun down at 4 °C, and 4,000xg
for 20min. The pellet was collected for further purification.

33
Chapter 6 Purification of pET44-GB3R2-His6-TEV-CD33(D140-
D298)-C169S
6.1 Methods
6.1.1 Lysis of cells
The collected cell pellets were resuspended in 30 ml per L culture volume of lysis buffer: 50 mM
Na2HPO4/NaH2PO4 pH 8.0, 300 mM NaCl (17.54 g/L), 20 mM imidazole (1.36 g/L), 100 mM SDS. Beta-
mercaptoethanol was added to the cell suspension to a final concentration of 2mM to break up inclusion
bodies. Then the cell suspension was sonicated, and spun down at 20C for 20min at room temperature.
Finally, the supernatant was filtered by filter paper with funnels, thus ready for loading.

6.1.2 IMAC column for getting His-tagged Protein of Interest
The purpose of this step is to obtain hexahistidine-tagged proteins from lysis.
After being re-charged with 5mL 0.1 M NiSO4 and equilibrated with wash buffer I, 50 mM
Na2HPO4/NaH2PO4 pH 8.0, 300 mM NaCl, 25 mM SDS, the filtered lysate supernatant was loaded onto the
IMAC column at 1.0 mL/min.
After washing with 30 column volumes(CV) of wash buffer I, 15CV of wash buffer II: 50 mM
Na2HPO4/NaH2PO4 pH 8.0, 300 mM NaCl (17.54 g/L), 8 M urea (480 g/L), 20 mM imidazole was used to
wash out SDS.
Wash buffer III, 50 mM Na2HPO4/NaH2PO4 pH 8.0, 300 mM NaCl, 8 M urea, 50 mM imidazole was also
used to wash out nonspecific binding protein.
Finally, the protein of interest was eluted by Elution buffer: 50 mM Na2HPO4/NaH2PO4 pH 8.0, 300 mM
NaCl, 8 M urea, 300 mM imidazole.

34
6.1.3 Dialysis and TEV cleavage
The aim is to remove the fusion tag—GB3R2(His6).
The eluate was dialyzed overnight against 5L of 50mM Tris pH8.0 via using a Spectra/Por 3 Dialysis
Membrane(MWCO 3500, 45mm,cat. No. S632724).
Usually, TEV was added to the dialyzed protein solution according to its concentration, to reach a final
molar ratio: TEV: Protein=1:50.
Also, the plan was also tried that TEV was added in the dialysis bag, co-refolding with the protein of interest
and thus start cleaving in the presence of around 2M Urea. This will be discussed in detail in the discussion
part. The protein of interest can be in both supernatant and pellet, depending on different situation. The
pellet can be re-dissolved with the lysis buffer.

6.1.4 IMAC column for obtaining cleaved Protein of Interest without His-tag
The purpose is to obtain protein of interest without a fusion protein tag(GB3R2). The protein of interest
cannot be bound in IMAC column and existed in the flowthrough.
The re-charging of Ni
2+
and equilibrium with wash buffer I was the same as described in 6.2. The cleaved
protein solution was loaded onto the IMAC column at 0.5mL/min. The flowthrough was collected
containing the cleaved protein of interest.
After loading and washing with wash buffer I, the bound proteins were eluted with Elution Buffer.

6.1.5 High Performance Liquid Chromatography(HPLC)
The reverse-phase HPLC was utilized in this study.
First, Hamilton PRP-3 (305X7.0mm, cat. no. 79468) was set at 60 °C, and the degasser was turned on.
Pumps were primed with A, 0.1% TFA in MilliQ-H2O and B, 0.1% TFA, 70% acetonitrile, 30% n-propanol
(1-propanol) using the program “prime_pumps_AA”. The loop was washed with 25mL MQ water.
35
During “Load AA” program, the sample, after being filtered with 0.20umilter, was injected. The program
“30_90_30min_3ml_AA” (gradient from 30 to 90% B in 30 min at 3 ml/min )was applied to purify the
proteins. Each peak was collected.
After finish purifying the protein, the programs “column_wash_lamp off” (from 100% MilliQ-H2O to 50%
acetonitrile, 50% n-propanol in 30 min and back, followed by pump water storage) and “store_pumps” for
storage in water were run.
The collected samples were frozen in liquid N2 for 10-15 min and freeze-dried. Dried power will be prepared
for NMR analysis in the future.

36
6.2 Results
6.2.1 Chromatography of IMAC column

Figure 16 IMAC chromatography of pET44-GB3R2-His6-TEV-CD33(D140-D298)-C169S
A peak was observed during elution. No other peaks appeared.

Peak-1
37

Figure 17 IMAC chromatography of cleaved pET44-GB3R2-His6-TEV-CD33(D140-D298)-C169S
The supernatant of TEV-cleaved protein of interested was purified in this step. CD33(D140-D298)-C169S should be in the peak-
1, GB3R2-His6 and TEV were eluted in Peak-2 theoretically.

Peak-1
Peak-2
38

6.2.2 SDS-PAGE of pET44-GB3R2-His6-TEV-CD33(D140-D298)-C169S

Figure 18 SDS-PAGE of pET44-GB3R2-His6-TEV-CD33(D140-D298)-C169S

1 2 3 4 5 6 7 8 9 10 11 12
1. Ladder
2. Loading Flowthrough of GB3R2-CD33-IgC2-TM
3. Wash Buffer I Flowthrough of GB3R2-CD33-IgC2-TM
4. Wash Buffer II Flowthrough of GB3R2-CD33-IgC2-TM
5. Wash Buffer III Flowthrough of GB3R2-CD33-IgC2-TM
6. Elution of GB3R2-CD33-IgC2-TM
7. Loading Flowthrough of KSI-CD33-IgC2-TM
8. Wash Buffer I Flowthrough of KSI-CD33-IgC2-TM
9. Wash Buffer II Flowthrough of KSI-CD33-IgC2-TM
10. Wash Buffer III Flowthrough of KSI-CD33-IgC2-TM
11. Elution of KSI-CD33-IgC2-TM
12. Supernatant of dialyzed GB3R2-CD33-IgC2-TM cleaved by TEV
24kDa
24kDa
72kDa
<15kDa
39

Figure 19 SDS-PAGE of TEV-cleaved pET44-GB3R2-His6-TEV-CD33(D140-D298)-C169S

1 2 3 4 5 6 7 8 9 10 11 12
18kDa
<15kDa
1.Ladder
2. Blank
3. Pellet (TEV-cleaved dialyzed protein)
4. Supernatant (TEV-cleaved dialyzed protein);W/O SDS
5. Supernatant (TEV-cleaved dialyzed protein);with 25mMSDS
6. IMAC column: FT
7: Elution: TEV, GB3R2-tag
8,9: Early peaks((Figure21 Peak1 half-1 and half-2) in HPLC
10: Blank
11: Main peak(Figure21 Peak2) half-1;
12: Main peak(Figure21 Peak2) half-2
24kDa
72kDa
40

Figure 20 SDS-PAGE of Pellet, Supernatant and Flowthrough(containing protein of interest) of pET44-GB3R2-His6-TEV-
CD33(D140-D298)-C169S
Lane-1: Ladder
Lane-2, 4, 6, 8, 12: Blank
Lane-3: Pellet from TEV-cleaved products with beta-ME
Lane-5: Supernatant from TEV-cleaved products with beta-ME
Lane-7: Flowthrough collected from IMAC column with beta-ME
Lane-9: Pellet from TEV-cleaved products without beta-ME
Lane-10: Supernatant from TEV-cleaved products without beta-ME
Lane-11: Flowthrough collected from IMAC column without beta-ME
Lane-12: Blank

1 2 3 4 5 6 7 8 9 10 11 12
24kDa
72kDa
18Kda
41
6.2.3 HPLC of pET44-GB3R2-His6-TEV-CD33(D140-D298)-C169S

Figure 21 FPLC of the purified product of cleaved pET44-GB3R2-His6-TEV-CD33(D140-D298)-C169S
The Peak-1 and Peak-2 were observed and collected. The earlier the peak comes out, the more hydrophilic it is. The peaks were
subsequently were frozen in liquid N2 for 10-15 min and freeze-dried. Dried power would be prepared for NMR analysis in the
future.

Peak-1
Peak-2
42
Chapter 7 Discussion
7.1 Expression and Purification of IgV-IgC2 domain(Construct-1,2,3)
IgV-IgC2 domain was previously tried to be expressed with alpha mating(aM) signal peptide by other lab
mates but no clear band was observed. This is the reason why we were looking for other endogenous signal
peptides in Pichia and finally chose Msb2 because of its generally better performance
11
.

From Figure 5, bands at 42 kDa were possibly glycosylated pPIC9K-Msb2-CD33(D18-T232)-
Flag(Construct-1). The molecular weight of this recombinant protein is ~25kDa. With 5 N-linked
glycosylation sites(100, 113, 160, 209 and 230) predicted from Uniprot, and estimating roughly the
molecular weight of each N-linked polysaccharide ~2.33kDa
14
, the estimated molecular weight of
pPIC9K-Msb2-CD33(D18-T232)-Flag is ~37kDa. The actual bands migrated as 42kDa( and lower faint
bands appeared less than 40kDa). This observation may be explained by hyper O-linked glycosylation in
Pichia pastoris
15
. This guess could be tested by using Endo Hf to cleave off N-linked glycosylation and
running the SDS-PAGE again. pPIC9K-Msb2-CD33(D18-T232)-Flag was expressed in 2 Liter and
purified by anion-exchange column(Q column), but it failed.

To make purification easier, we designed pPIC9K-Msb2-CD33(D18-T232)-Flag-His6(Construct-2) and
pPIC9K-Msb2-CD33(D18-T232)-Flag-His10(Construct-3). The hexahistidine tag(His6) was designed for
HiTrap IMAC column, with a loading flow rate at most 1mL/min; the decahistidine tag(His10) was
designed for HiTrap Excel column, with a loading flow rate 5mL/min. From Figure 6(a) and (b), bands at
42kDa could be observed in both Western Blotting. However, the bands were faint when His6-tag had been
attached, and the bands were almost invisible when His10-tag had been attached. Although the condition
of each Western blotting may vary, Figure 5 and Figure 6 were adjusted to the similar brightness for
comparison.

43
The relative expression level of pPIC9K-Msb2-CD33(D18-T232)-Flag-His6(Construct-2) is still
acceptable, so we conducted 2 Liter culture expression. The results were not satisfying, thus we were
looking for improvement for Pichia culture. Detergent Tween-20 had been tested in some pilot experiments
of IgC2-Linker domain, and better results were obtained in purification process. At this stage, solubilization
ability was considered the dominant reason for improvement of protein expression, besides foam
stabilization and gas exchange efficiency. The details were discussed in the part of IgC2-Linker domain.

Tween-20(0.2 g/L) was added to the Pichia pastoris culture during induction
12,13
. The Critical Micelle
Concentration (CMC) of Tween-20 is 0.08mM(0.042g/L), and 0.2 g/L was applied in the induction step,
which means 0.158g Tween-20 formed micelles per liter.

As shown in Figure 14, only one high peak(Peak-1) was observed during washing with Wash buffer
I(20mM imidazole) and no elution peak was seen. The Peak-1 was used as samples for SDS-PAGE in
Figure 15(Lane-6 with beta-ME; Lane-11 without beta-ME), but no band was seen, possibly due to low
expression concentration of protein of interest.

In addition, the binding to polysaccharide ability of Ig-V domain may be a reasonable cause of low
expression of this protein because cell walls of Pichia pastoris are composed of polysaccharide, glycolipid
and glycoproteins. The expressed protein with Ig-V domain may be bound to these cell walls and cell wall
debris, thus being trapped in the precipitation.

As mentioned previously, the application of Tween-20 has small improvement for pPic9K-alphaMATING-
KREAEA-Flag-CD33(D140-G266)-C169S-His6(Construct-4) in some pilot experiments, so we continue
the purification with it.

44
7.2 Expression and Purification of IgC2-Linker domain(Construct-4, 5)
pPic9K-alphaMATING-KREAEA-Flag-CD33(D140-G266)-C169S-His6(Construct-4) was done by
Technician Alan Situ previously. The expression level seems good(Figure 8), but purification of it was
unsatisfying.

The addition of Tween-20 in the process of Methanol induction contributed to obtaining a single peak
containing relatively pure polyhistidine-tagged overexpressed proteins in the elution process. Undesired
proteins, polysaccharides, cell wall debris, other impurities, and a small proportion of desired proteins were
possibly trapped in the micelles, thus their interaction with overexpressed protein of interest(IgC2-Linker)
was undermined and they were less possible to be trapped in IMAC column. Compared with undesired
proteins, overexpressed protein of interest(IgC2+Linker) was supposed to have higher concentration, so
loss of a small part of recombinant protein IgC2+Linker would be acceptable and majority of it could be
obtained from IMAC column eluate.

Foam is unavoidable in yeast culture because of gas exchange, turbulence and foam-promoting substances
in culture broth. The foaming can undermine the gas exchange efficiency at the gas/liquid surface in the
flask
16,17
, and Pichia pastoris requires enough oxygen to grow. Antifoam agents could be helpful, but
whether antifoam agents could potentially cause stress response and impair overexpression remains
unknown. If possible, antifoam agents are worth studying in the future. Currently, using detergent to
improve expression and purification is preferred to be tested singly at this stage. Appropriate solubilization
is the prior aim, and foaming situation is the secondary purpose.

For solubilization, critical micelle concentration(CMC) is one of the important and operable variable. Ion
strength does not significantly influence the CMC of nonionic detergent like Tween-20. Temperature is
vital for nonionic detergent, but the temperature range for Pichia pastoris culture will not change greatly.
Since the culture condition has been optimized, the temperature will be kept at 27 °C and other composition
45
will not changed. Thus, only detergent and its concentration will be changed. In addition, the solubilization
is not as stronger as better. Too strong solubilization will trap too much protein of interest into micelles
leading to a lower amount of protein of interest from IMAC eluate. Thus, the concentration which is a little
higher than the CMC to form appropriate amount of micelles could perform well theoretically.

Multiple factors such as temperature, ion strength, protein concentration, yeast density, detergent
concentration, detergent CMC, shaking RPM, and etc. can potentially change foaming situation, so it might
be impossible to predict foaming situation in advance. To some extent, a concentration higher than CMC
can make the surface tension of culturing media relatively stable, thus providing a comparatively stable
environment for Pichia pastoris. Without a considerable concentration of detergent, the surface tension will
change unpredictably along with yeast growth. Although little is studied about surface tension condition
with yeast culture performance, a more constant environment can avoid potential accidents.

As mentioned before, the appropriate solubilization and purification improvement are prior to foaming
situation. We aimed to confirm a detergent with better performance as well as its usage concentration at
first, and then the foaming situation would be checked empirically.

Nonidet P-40 subsitute usually forms less foam during shaking empirically, and nonidet P-40 can solubilize
membrane protein
18
while some receptors on cell wall debris may interfere with our protein of interest. Its
CMC is 0.25mM (~0.154g/L) while 0.2g/L was still used during Methanol induction. Only around 0.046 g
Nonidet P-40 substitute could form micelles, which is not too much compared with Tween-20. In pilot
experiments, 0.2g/L Nonidet P-40 substitute performed well. Therefore, P-40(0.2g/L) was added to the
Pichia pastoris culturing media when expressing this recombinant protein.

From Figure 10, a high peak during elution process could be observed, and the subsequent purification
indicated that more recombinant proteins were obtained due to the addition of P-40(0.2g/L).
46
Furthermore, we planed to test whether the addition of Endo Hf during the last 4 hours in the culture media
could simplify the purification process. From Figure 10, Figure 11 and Figure 12, almost no obvious
difference was observed about the peak of our protein of interest, and the molecular weight o each peak
was tested in the SDS-PAGE(Figure 13). The additional peak(Peak-1) in Figure 12(a) was not protein,
which has no protein-related signal during solution-state NMR analysis. The calculation depending on the
Ve(volume of elution), and the result greatly indicated that IgC2-Linker recombinant protein exists as
dimmers under the condition of pH=7.4, 150mM NaCl.

The concentration of purified Construct-4 was lower in the presence of Endo Hf in the last four hours during
culturing. This could be the reason of loss of glycosylation because glycosylation can enhance the solubility
of protein possibly due to increasing amounts of possible interactions between the surface of glycoprotein
and e surrounding solvent molecules
19
. When de-glycosylated, the protein may be less soluble and less
dissolved in the culture media.

In Figure 15, every lane was from a peak obtained in each chromatography. Bands shifting as 22-25kDa
should be glycosylated IgC2-Linker(pPic9K-alphaMATING-KREAEA-Flag-CD33(D140-G266)-C169S-
His6), with three N-glycosylation sites(160, 209 and 230) predicted by Uniprot, estimating that each N-
glycosylation contributes 2.33kDa
14
to the protein molecular weight. The theoretical molecular weight of
non-glycosylated protein(IgC2-Linker; pPic9K-alphaMATING-KREAEA-Flag-CD33(D140-G266)-
C169S-His6) should be around 15kDa as a monomer. In Table 1 and 2, the molecular weight of the final
results were calculated, showing that IgC2-Liner domains could exist as dimers under the condition of
25mM Sodium Phosphate buffer, 150mM NaCl. The results of these bands and calculation are considerably
close to the theoretical value.

Generally, the whole process of expression and purification of pPic9K-alphaMATING-KREAEA-Flag-
CD33(D140-G266)-C169S-His6(Construct-4) is relatively successful, and enough amount of this
47
recombinant protein of high quality can be obtained, as well as more improvement and optimization will
be done for IgC2-Linker recombinant.

7.3 Expression and Purification of IgC2-TM domain(Construct-6)
IgC2-TM(pET44-GB3R2-His6-TEV-CD33(D140-D298)-C169S) recombinant protein was expressed in
E.coli, different from other constructs. GB3R2 was fused in order to improve the solubility of IgC2-TM
domain via a linker which could be cleaved by TEV. After TEV cleavage, some precipitate was visible. At
the beginning, it was difficult to predict whether the cleaved protein of interest existed in precipitates or
supernatant. This is because the IgC2 domain is relatively more hydrophilic and the TM domain is more
hydrophobic, but it is not possible to judge its overall effect on solubility. Due to the limitation of time, the
supernatant of TEV cleaved products was purified by IMAC column and HPLC in this step. The pellet will
be dissolved and purified in the future.

From the chromatography(Figure 16, peak-1) and SDS-PAGE(Figure 18, Lane-6), a 24kDa band could be
observed. After cleavage of TEV, both the supernatant and re-dissolved pellet were tested by SDS-PAGE.
From Figure 19 and Figure 20, bands at 18 kDa were believed to be cleaved protein(IgC2-TM) while bands
around 10kDa(less than 15kDa) were believed to be GB3R2 fusion part. From HPLC(Figure 21), although
Peak-1 and Peak-2 were observed, only Peak-2 was possible to be the cleaved protein(IgC2-TM) by SDS-
PAGE(Figure 19; Lane 11, 12) because reasonable bands were observed(18kDa as monomer; other bands
were possibly dimers, trimers, etc.). Both Peak-1 and Peak-2 collected from HPLC were freeze-dried at
first, and then a small amount of these powder was re-dissolved with the lysis buffer. This is just a simple
pre-test. If there is plenty of time and protein concentration is enough, it would be much more suitable to
prepare these powder as NMR sample and conduct NMR analysis to obtain more information.

The expression and purification of IgC2-TM are relatively successful as well. The results indicates that it
is not satisfying to add TEV during dialysis. Next time, TEV will be added after dialysis as usual, instead
48
of co-refolding with recombinant protein. More improvement will be made in the aspect of TEV cutting
because of the incomplete cleavage. In addition, the accurate amount of the cleaved protein of interest(IgC2-
TM) from pellets will be confirmed. Although thicker bands could be observed in SDS-PAGE(Figure 20;
Lane-3), pellets are re-dissolved in lysis buffer of smaller volume thus making it impossible and
unreasonable to compare the bands from SDS-PAGE in order to estimate the amount of protein of
interest(IgC2-TM). Therefore, it will be more accurate to purify both supernatant and pellet from TEV-
cleaved protein with IMAC column and HPLC, and then their concentration can be measured by UV-
spectroscopy after re-dissolving their freeze-dried powder. If more proteins are obtained in pellets, next
time we will focus on pellets when expressing isotope-labelled IgC2-TM.

49
Chapter 8 Summary and Conclusion
Human CD33 is a membrane protein expressed on microglia in the brain, which is associated with
Alzheimer’s disease by acting as a molecular mediator. In this study, we are trying to express isotope-
labeled extracellular domain of CD33(IgV-IgC2; IgC2-Linker; IgC2-TM) in order to prepare NMR samples
for further solution-state NMR analysis.

The expression and purification of IgC2-Linker(Construct-4) and IgC2-TM(Construct-6) appeared to be
promising. 15-N labeled IgC2-Linker has been already expressed and purified. For IgC2-Linker
domain(pPic9K-alphaMATING-KREAEA-Flag-CD33(D140-G266)-C169S-His6), molecular weight of
non-glycosylated IgC2-Linker should be 15kDa as a monomer, and glycosylated molecular weight should
be 22-25kDa. For IgC2-TM domain(pET44-GB3R2-His6-TEV-CD33(D140-D298)-C169S), the molecular
weight before cleavage should be 25.5kDa, and the molecular weight of CD33(D140-D298) should be
18kDa after cleavage. The IgC2-Linker domain exists as dimmers under the condition of pH=7.4, 150mM
NaCl, and the glycosylation appeared not play an important role in the dimerization. Detergent Nonidet P-
40 substitute(0.2g/L) worked well in IgC2-Linker project. Appropriate solubilization ability may contribute
to improvement of expression and purification in Pichia pastoris. Further improvement will be made to
these two domains to obtain more isotope-labeled recombinant protein of these two domains.

The effort to express IgV-IgC2 domain by replacing alpha mating factor signal peptide with a native signal
peptide sequences called Msb2 seems effective to some extent. The binding affinity nature of IgV domain
may make the expression and purification difficult and complicated. The detergent Nonidet P-40 substitute
will be applied in the culturing of IgV-IgC2 domain in the future. Also, the mutation of the binding site
119(R) will be done to test whether this may be the reason. In the future, R119A mutation will be made to
test whether the binding ability undermine the expression of IgV domain of CD33 in Pichia pastoris.

50
References

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8 Freeman, S. D., Kelm, S. r., Barber, E. K. & Crocker, P. R. Characterization of CD33 as
a New Member of the Sialoadhesin Family of Cellular Interaction Molecules. Blood 85,
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9 Malik, M. et al. CD33 Alzheimer's risk-altering polymorphism, CD33 expression, and
exon 2 splicing. J Neurosci 33, 13320-13325, doi:10.1523/JNEUROSCI.1224-13.2013
(2013).
10 Naj, A. C. et al. Common variants at MS4A4/MS4A6E, CD2AP, CD33 and EPHA1 are
associated with late-onset Alzheimer's disease. Nat Genet 43, 436-441,
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11 Duan, G. et al. Screening endogenous signal peptides and protein folding factors to
promote the secretory expression of heterologous proteins in Pichia pastoris. J Biotechnol
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laccase Lcc9 expressed in Pichia pastoris. Journal of Bioscience and Bioengineering 128,
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Abstract (if available)

Abstract Alzheimer’s Disease (AD) is affecting millions of people worldwide. Innate immune response and inflammation mediated by microglia and astrocytes may play a role in AD according to recent studies. CD33 is expressed on the membrane of microglia in the brain, composed of extracellular domain(IgV and IgC2), a single-pass transmembrane domain(TM) and a cytosolic domain(CS). Genome-wide association studies(GWAS) show that CD33 is one of the genes associated with AD. ? However, little is known about the real structure and function of CD33 in microglia up to now. In this study, extracellular domain of human CD33 was both expressed in E.coli and Pichia pastoris. The aim is to express isotope-labeled IgV-IgC2, IgC2-linker and IgC2-TM domain for further solution-state NMR analysis. ? To date, 15-N labeled IgC2-Linker recombinant protein was successfully expressed and purified. The optimization of culturing condition and addition of detergent P-40(nonidet P-40 substitute) can improve the expression level of IgC2-Linker in Pichia pastoris as well as purification process. The IgC2-TM recombinant protein was also expressed and purified in E.coli. The expression and purification results for IgV-IgC2 domain was not satisfying. The reason could be the IgV domain’s binding affinity to polysaccharide, glycoprotein and glycolipid. It is promising to produce isotope-labeled IgC2-Linker and IgC2-TM with these improvements and constructs.

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Asset Metadata

Creator Dai, Xuhang (author)

Core Title Expression and purification of extracellular domain of human CD33 in Escherichia coli and Pichia pastoris

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Medicine

Degree Conferral Date 2021-08

Publication Date 07/22/2021

Defense Date 06/21/2021

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag Alzheimer's disease,CD33,detergent,Escherichia coli,glycoprotein,microglia,OAI-PMH Harvest,Pichia pastoris,protein purification,receptor,transmembrane protein

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Advisor Ulmer, Tobias (committee chair), Langen, Ralf (committee member), Siemer, Ansgar (committee member)

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