Production of functional single-chain variable fragment specific for food-born mycotoxin, aflatoxin B1 in engineered Escherichia coli

(1)

저작자표시-비영리-변경금지 2.0 대한민국 이용자는 아래의 조건을 따르는 경우에 한하여 자유롭게 l 이 저작물을 복제, 배포, 전송, 전시, 공연 및 방송할 수 있습니다. 다음과 같은 조건을 따라야 합니다: l 귀하는, 이 저작물의 재이용이나 배포의 경우, 이 저작물에 적용된 이용허락조건 을 명확하게 나타내어야 합니다. l 저작권자로부터 별도의 허가를 받으면 이러한 조건들은 적용되지 않습니다. 저작권법에 따른 이용자의 권리는 위의 내용에 의하여 영향을 받지 않습니다. 이것은 이용허락규약(Legal Code)을 이해하기 쉽게 요약한 것입니다. Disclaimer 저작자표시. 귀하는 원저작자를 표시하여야 합니다. 비영리. 귀하는 이 저작물을 영리 목적으로 이용할 수 없습니다. 변경금지. 귀하는 이 저작물을 개작, 변형 또는 가공할 수 없습니다.

(2)

A Thesis for the Degree of Master of Science

Production of functional single-chain variable fragment

specific for food-born mycotoxin, aflatoxin B

1

in engineered Escherichia coli

재조합

대장균에서

곰팡이

독소 아플라톡신

B1에

특이적인

재조합

항체의 생산

By

Hyo-Ran Lee

School of Agricultural Biotechnology

Seoul National University

(3)

A Thesis for the Degree of Master of Science

Production of functional single chain variable fragment

specific for food-born mycotoxin, aflatoxin B

1

in engineered Escherichia coli

Advisor: Professor Jin-Ho Seo

Submitted in Partial Fulfillment of the Requirements

for the Degree of Master of Science

By

Hyo-Ran Lee

School of Agricultural Biotechnology

Seoul National University

(4)

農學碩士學位論文

Production of functional single chain variable fragment

specific for food-born mycotoxin, aflatoxin B

1

in engineered Escherichia coli

재조합

대장균에서

곰팡이

독소 아플라톡신

B1에

특이적인

재조합

항체의 생산

指導敎授 徐鎭浩 이 論文을 農學碩士學位論文으로 提出함 2014年 2月 서울大學校 大學院 農生命工學部食品生命工學專攻李孝爛李孝爛 의 農學碩士學位論文을 認准함 2014年 2月 委員長 유 상 렬 副委員長 서 진 호 委員 최 영 진

(5)

학위논문 원문제공 서비스에 대한 동의서 본인은 본인의 연구결과인 학위논문이 앞으로 우리나라의 학문발전에 기여할 수 있도록, 서울대학교 중앙도서관을 통한 “학위논문 원문제공 서비스”에서 다음과 같은 방법 및 조건하에 논문을 제공함에 동의합니다. 1. 인터넷을 통한 온라인 서비스와 보존을 위하여 저작물의 내용을 변경하지 않는 범위내에서의 복제를 허용함. 2. 저작물을 PDF로 구축하여 인터넷을 포함한 정보통신망에서 공개 하여 논문 일부 또는 전부의 복제(배포 및 전송에 동의함. 3. 해당 저작물의 저작권을 타인에게 양도하거나 또는 출판허락을 하였을 경우 1개월이내에 서울대학교 중앙도서관에 알림. 4. 배포, 전송된 학위논문은 이용자가 다시 복제 및 전송할 수 없으 며 이용자가 연구목적이 아닌 상업적 용도로 사용하는 것을 금함 에 동의함. 2014 년 2 월 일 서울대학교총장 귀하 ---

논문제목 : Production of functional single chain variable fragment specific for a

food-born mycotoxin, alfatoxin B1 in engineered Escherichia coli

학위수여 : R 석사 £ 박사 학 과 : 農生命工學部 학 번 : 2012-21177 연 락 처 :

(6)

i

ABSTRACT

Aflatoxin B1 (AFB1) is a secondary fungal metabolite produced by Aspergillus flavus and A. parasiticus. International Agency of Research on Cancer (IARC) classified AFB1 into a group I carcinogen for humans. Aflatoxin B1 contaminate grains and crops that human and livestock consume. Detection of aflatoxin B1 is an essential step to reduce this threat. Three mtehods have been used to detect aflatoxin B1; biological, analytical, and immunological methods. Enzyme immunosorbent assay (ELISA) is highly specific, sensitive, simple and rapid for measuring aflatoxins in foods. In previous research, scFv was cloned from the murine monoclonal antibody to aflatoxin B1 and produced in E. coli. However, the scFv was expressed in insoluble form, so an in vitro refolding procedure was necessary to acquire soluble and active scFv. In the following previous research, by co-expressing several folding modulators from E. coli and Bacillus subtilis with scFv in E coli, soluble scFv was produced and secreted into the periplasm of E. coli. Even though soluble scFv was produced, the proportion of the soluble scFv in the total expression level was low. Thus, a new expression system needs to be developed to produce soluble scFv in large proportion.

To increase the proportion of soluble scFv, maltose binding protein (MBP) fusion, codon optimization, and new E. coli C41(DE3) strain used as a host were introduced.

First, MBP fusion with the scFv gene for enhancing an expression level in soluble form of scFv was attempted. A MBP was fused with the N or C-terminal of scFv. An expression level of the fused scFv slightly increased due to the MBP fusion. An expression level of the C-terminal MBP fused scFv was higher than N-terminal fusion. However, the expression form of scFv

(7)

ii

was still insoluble.

Codon optimization of the scFv gene was adopted. Elimination of codon usage bias makes the heterologous protein expression easier. The codon optimized scFv fused with MBP were expressed in E. coli BL21(DE3). Despite codon optimization, it was not effective to produce in soluble form. Thirdly, besides BL21(DE3), other E. coli host strains were selected to express scFv. C41(DE3) and Origami (DE3) strains were employed to express the MBP-fused scFv.

The C41(DE3) strain carrying the scFv fused N-terminal MBP was able to produce soluble and active scFv with the high proportion of the total expression scFv.

A fed-batch fermentation was conducted to maximize the production of the MBP. The maximum concentration of scFv was 810 mg/L. The scFv-MBP fusion protein was purified with affinity chromatography using hisitidine tags for characterizing the antibody properties.

The produced scFv was tested for antigen-binding activity by indirect ELISA. The antigen-binding activity was determined by the absorbance of the MBP-fused scFv being proportional to concentrations aflatoxin B1. The secondary structure of the scFv protein was analyzed by circular dichroism in range of 190 ~ 250 nm. The beta sheet structure was identified as the secondary structure of scFv.

The new expression system consisting of the C41(DE3) strain carrying the scFv fused N-terminal MBP with the signal sequence of MBP was applied to other types of scFv such as fumonisin B1 and deoxynivalenol. The two scFv of fumonisin B1 and deoxynivalenol were successfully expressed in soluble form. In conclusion, this thesis allowed the development of a novel expression system for soluble production of various scFv in E. coli.

(8)

iii

Keywords: Escherichia coli, scFv, fusion protein, Afaltoxin B1, protein

expression system

Student Number: 2012-21177

LIST OF TABLES

Table 1. Antibody classes ··· 9

Table 2. Rarely used codons in E. coli ··· 10

Table 3. Generally used solubility-enghancing fusion partners ··· 12

Table 4. Sequence of the primers used in this research··· 29

(13)

viii

LIST OF FIGURES

Figure 1. Metabolic conversion of aflatoxin B1 and following DNA addcuct formation mutating hepatocyte DNA to carcinogenesis ···7 Figure 2. Subunit composition and domain distribution of immunoglobulin

and single-chain variable fragment (scFv) antibody ··· 8 Figure 3. Protein expression using solubility tags ··· 11 Figure 4. Toxic protein such as green fluorescent protein or red fluorescence

inducing protein expressed in C41 and BL21 ··· 13 Figure 5. Standard curve of secondary structure; alpha-helix, beta sheet, and

random coil by circular dichroism ··· 14 Figure 6 Amino acid sequence of AFB1 scFv and MBP fusion protein 31 Figure 7. Amino acid sequence of fumonisin B1 scFv with 6 histidine tag

··· 32 Figure 8. Amino acid sequence of deoxynivalenol scFv with 6 histidine tag

··· 32 Figure 9. Codon usage table of non-codon optimized scFv gene in E. coli

··· 33

(14)

ix

Figure 10. Codon usage table of codon optimized scFv gene in E. coli 34 Figure 11. Purification process ··· 35 Figure 12. Diagram of indirect ELISA ··· 36 Figure 13. Genetic map of non-codon optimized scFv gene for aflatoxin B1 ··· 43 Figure 14. Genetic map of codon optimized scFv gene with signal sequence for aflatoxin B1 ··· 44 Figure 15. Genetic map of codon optimized scFv gene without signal sequence for aflatoxin B1 ··· 45 Figure 16. Genetic map of fumonisin B1 and deoxynivalenol scFv gene with signal sequence for aflatoxin B1 ··· 46 Figure 17. Expression of non-MBP fusion scFv in BL21(DE3) at 37°C and 0.2 mM IPTG. ··· 48 Figure 18. Expression of MBP fusion scFv in BL21(DE3) at 37°C and 0.2 mM IPTG. ··· 49 Figure 19. Expression of MBP fusion codon optimized scFv with signal sequence in BL21(DE3) at 37°C and 0.2 mM IPTG. ··· 50

(15)

x

Figure 20 Expression of MBP fusion codon optimized scFv without signal sequence in BL21(DE3) at 37°C and 0.2 mM IPTG. ··· 51 Figure 21. Expression of MBP fusion scFv in C41(DE3) at 37°C and 0.2 mM IPTG ··· 52 Figure 22. Expression of MBP fusion codon optimized scFv with signal sequence in C41(DE3) at 37°C and 0.2 mM IPTG. ··· 53 Figure 23. Expression of MBP fusion codon optimized scFv without signal sequence in C41(DE3) at 37°C and 0.2 mM IPTG. ··· 54 Figure 24 Expression of MBP fusion codon optimized scFv without signal sequence in Origami (DE3) at 37°C and 0.2 mM IPTG··· 55 Figure 25. Profile of fed-batch fermentation ··· 56 Figure 26. Purification of scFv and MBP fuison protein ··· 57 Figure 27 Antigen (aflatoxin B1) binding activity of non-codon optimized scFv and MBP fuison protein by indirect ELISA ··· 58 Figure 28. Antigen (aflatoxin B1) binding activity of codon optimized scFv and MBP fuison protein by indirect ELISA ··· 59 Figure 29. Color reaction of substrate to measure antigen binding activity by indirect ELISA ··· 60

(16)

xi

Figure 30. Factor Xa protease treatment to take scFv itself ··· 61 Figure 31. Secondary structure analysis of scFv by circular dichroism 62 Figure 32. Expression of MBP fusion scFv of fumonisin B1 in C41(DE3) at 37°C and 0.2 mM IPTG. ··· 63 Figure 33. Expression of MBP fusion scFv of deoxynivalenol in C41(DE3) at 37°C and 0.2 mM IPTG. ··· 64

(17)

1

I. INTRODUCTION

1. A

FLATOXIN

B

1

Aflatoxin B1 (AFB1) is a secondary fungal metabolite produced by Aspergillus flavus and A. parasiticus. AFB1 in dry state is very stable to heat up to the melting point, 269℃ (Moghaddam et al., 2001). It possesses hepatotoxic and mutagenic properties by damaging hepatocyte DNA and causes toxic hepatitis, hemorrhage, immunosuppression and hepatic carcinoma (Reddy et al., 2009) (Fig. 1). International Agency of Research on Cancer (IARC) classified AFB1 into a group I human carcinogen for humans (IARC (International Agency for Research on Cancer)). KFDA (Korea Food & Drug Administration) regulate maximum limits in grains, beans, peanut, nuts, and their processed food (grinding, cutting etc.) as 10 μg/kg (KFDA: Revision of food standards (KFDA notification 2007-63) (2007)).

2. Detection methods of AFB

1

Aflatoxin B1 contamination in grain poses a great threat to human and livestock health as well as international trade. Confirmation of the extent of contamination is the essential first step in reducing exposure to aflatoxin B1.

(18)

2

Detecting of aflatoxin B1 is necessary in a variety of situations ranging from in the field to in controlled laboratory settings. Accordingly, the technologies being developed range from those which can be conducted rapidly with minimal technical expertise, such as immunoassays and biosensors, to those which can be conducted by technical personnel 〔www.ncaur.usda.gov〕. For detecting aflatoxin B1, three main types of assays have been developed. These include biological, analytical and immunological methods. Immunological methods such as enzyme-linked immunosorbent assay (ELISA) are highly specific antibody-based and rapid tests for measuring aflatoxins in food. Furthermore, these methods are relatively simple to execute and analyze many samples at a time, yet are sensitive.

3. Single-chain variable region fragment antibody (scFv)

Single chain variable region fragment antibody (scFv) is a fusion protein of the variable region of the heavy and light chains (VH and VL) of immunoglobin, connected with a short linker peptide of amino acids. Antibody, also known as an immunoglobulin is a Y-shaped protein produced by B-cell that is used by the immune system to identify and neutralized antigen recognized as foreign objects such as bacteria and viruses. Antibody

(19)

3

molecules have a common structure of two identical light and heavy chains (Fig. 2). Each chain is divided into two regions, the variable (V) and constant (C) regions. In V region, the first 110 or so amino acids are of increased variability called complementarity-determining regions (CDRs). The CDRs of the light and heavy chains constitute the antigen-binding sites. Variable domains have β-sheet secondary structure. Heavy chains divide antibodies into five classes called IgG, IgA, IgM, IgD, and IgE (Table 1). Light chains classify antibodies into κ and λ chains. IgG is predominant immunoglobulin in humans with high specificity and affinity. Compared to IgG, scFv is small molecule and easy to produce. Up to date, scFv expressed in various systems such as mammalian cell, yeast, bacteria, plant, and also insect cells (Ho et al. 2006, Galeffi et al., 2006, Choo et al., 2002). scFv can be expressed in correctly folded and active form or aggregated form requiring in vitro refolding to make it active (Min et al., 2011). Bacterial expression system is most often applied for the production of scFv compared to the various expression system available (Ahmad et al., 2012).

4. Codon optimization

Heterologous protein production in E. coli is preferred expression system. E. coli expression system is beneficial in aspects of fast growth rate,

(20)

4

inexpensive media, and thoroughly understood DNA techniques (Makrides, 1996). Despite all benefits, heterologous protein production in E. coli may be decreased due to codon bias. Codon usage bias is differences in the frequency of occurrence of synonymous codons in coding DNA (Ermolaeva, 2001). Improved heterologous expression of genes may be obtained by replacing rare to more commonly used codons, codon optimization. Though, codon substitution in large scale may have influence on protein expression in the other ways which are not always anticipated (Wu et al., 2004).

5. Affinity tags

Expression of soluble protein in E. coli is major bottleneck in heterologous protein production. The most biochemically valuable proteins such as kinases, phosphatase, and many other enzymes, are convoluted to produce as soluble protein in E. coli. The rate of translation and protein folding in E. coli is faster than that in eukaryotic systems. A fast rate of protein synthesis and a slow rate of protein folding in E. coli may make protein insoluble and aggregate (Widmann et al., 2000). Changing some of the expression conditions such as reduced temperature or induction condition to enhance soluble protein production can be minimal (Kataeva et al., 2005). It appeared that some affinity tags could improve the solubility of some of the

(21)

5

companion proteins to which they were fused (Kapust et al., 1999 & Nygren et al., 1994) (Fig. 3). There are numbers of commonly used solubility-enhancing fusion tags that can be applied to express protein in E. coli (Table 2). Maltose binding protein (MBP) from E. coli is one of the most well-understood solubility factors and has a significant evidence that MBP fusions is able to express soluble proteins as the unfused proteins are insoluble. N-terminal fusion partners are preferred to C-N-terminal fusion (Dyson et al., 2004). Fusion tags such as histidine tag, polycationic amino acids tails are used for protein purification as well (Kweon et al., 2002). 10 arginine tag as polycationic amino acids tags like the preceding enhance soluble expression of Candida antarctica lipase B in E. coli (Jung et al., 2011). 6-lysine tagged ubiquitin fusion enhanced expression level of target protein (Kim et al., 2011).

6. E. coli strains for protein expression

In expression of heterologous protein, E. coli is eminent host organism due to the simplicity of its use, the high levels of expression commonly obtained, and the huge variety of expression plasmids that is available (Grisshammer et al., 1995). E. coli host strain BL21(DE3) is commonly used for the overexpression of both prokaryotic and eukaryotic proteins (Studier et al.,

(22)

6

1990). Frequently, overproduction of proteins cannot be attained due to the toxicity of the aimed protein causing bacterial cell death, despite a number of proteins have been produced with success to very high levels in BL21(DE3). Two mutant strains named C41(DE3) from BL21(DE3) are able to grow and continue to produce proteins at an elevated level (Miroux et al., 1996). The C41(DE3) strains have been applied to produce proteins that were expressed in low level in BL21(DE3) (Sorenson et al., 2003) (Fig. 4). Origami host strains that have mutations in both the thioredoxin reductase (trxB) and glutathione reductase (gor) genes enhance formation of disulfide bond in the cytoplasm (Bessette et al., 1999)

7. Circular Dichroism

Circular dichroism (CD) spectroscopy measures differences in the absorption of left-handed polarized light versus right-handed polarized light which arise because of structural asymmetry. CD determines whether a protein is folded and characterizes protein’s secondary structure, tertiary structure. Secondary structure can be measured by CD in the far-UV spectral region (190 - 250 nm). At these wavelengths, the chromophore is the peptide bond. Alpha-helix, beta-sheet, and random coil structures each give rise to a characteristic shape and magnitude of CD spectrum (Fig. 5). The CD signal

(23)

7

reflects an average of the entire molecular population. The DC spectrum of a protein in the near-UV (250 – 350 nm) can be characterized tertiary structure of protein. At these wavelengths, the chromophores are the aromatic amino acids and disulfide bonds. The CD signals they produce are susceptible to the overall tertiary structure of the protein.

(24)

8

Fig. 1. Metabolic conversion of aflatoxin B1 and following DNA adduct formation mutating hepatocyte DNA to carcinogenesis

(25)

9

Fig. 2. Subunit composition and domain distribution of immunoglobulin and single-chain variable fragment (scFv) antibody

(26)

10

Table 1. Antibody classes

Charateristic/Fuction IgG IgA IgM IgD IgE

Molecular Weight (KDa) 150 400 900 180 190

Heavy chain Γ α Μ Δ ε

Number of subunits 1 2 5 1 1

Serum level (㎍/㎖) 13.5 3.5 1.5 0.03 0.003

Allotypes G1,2,3,4 A1,2

(27)

11

Table 2. Rarely used codons in E. coli (Dominic et al., 2006)

Amino acid Rare codon(s)

Arginine AGG, AGA, CGG, CGA

Leucine CUA, CUC

Isoleucine AUA

Serine UCG, UCA, AGU, UCC

Glycine GGA, GGG

Proline CCC, CCU, CCA

(28)

12

Fig. 3. Protein expression using solubility tags (Dominic et al., 2006)

(29)

13

Table 3. Generally used solubility-enhancing fusion partners

Tag Source organism

MBP (Maltose-binding protein) Escherichia coli GST (Glutathione-S-trasferase) Schistosoma japonicum

Trx (Thioredoxin) Escherichia coli

NusA (N-Utilization substance) Escherichia coli SUMO (Small Ubiquitin-modifier) Homo sapiens

SET (Solubility-enhancing tag) Synthetic DsbC (Disulfide bond C) Escherichia coli Skp (Seventeen kilodalton protein) Escherichia coli T7 PK (Phage T7 protein kinase) Bacteriophage T7

GB1 (Protein G B1 domain) Streptococcus sp. ZZ (Protein A IgG ZZ repeat domain) Staphylococcus aureus

(30)

14

Fig. 4. Toxic protein such as green fluorescent protein (top) or red fluorescence inducing protein (bottom) expressed in C41 and BL21

(31)

15

Fig. 5. Standard curve of secondary structure; alpha-helix, beta sheet, and random coil by circular dichroism

(32)

16

7. O

BJECTIVES OF THE THESIS

This study is focused on the production of single chain variable fragment (scFv) in active form in engineered Escherichia coli. The specific objectives of this research were as follows:

1) Evaluation of factors such as codon optimization, maltose binding protein affinity tags, or E. coli strains affecting soluble expression of aflatoxin B1 scFv.

2) Applications of the expression strategies to other types of scFv. 3) Analysis of the physico-chemical and immunological properties

(33)

17

II. MATERIALS AND METHODS

1. Plasmids and strains

1.1 Enzymes and reagents

AFB1 conjugated to bovine serum albumin (AFB1-BSA conjugate) was bought from Sigma Aldrich Co. (St. Louis, MO, USA). Restriction endonucleases, T4 DNA ligase, Taq polymerase, Klenow fragment, and calf intestinal alkaline phosphatase (CIP) were purchased from New England Biolabs (Beverly, MA). Pwo DNA polymerase and dNTPs were from Hoffmann-La Roche (Basel, Switzerland). Phosphate buffered saline (PBS: 0.01 M phosphate buffer with 0.138 M NaCl, 0.0027 M KCl), phosphate buffered saline tween 20 (PBST: 0.01 M phosphate buffer with 0.138 M NaCl, 0.0027 M KCl, 0.05 % Tween 20), carbonate-bicarbonate buffer capsules (0.05M carbonate-bicarbonate buffer, pH 9.6), bovine serum albumin (BSA), phosphate-citrate buffer tablets (0.05M phosphate-citrate buffer, pH 5.0, 1 tablet/100 ml), 5'-tetramethyl benzidine dihydrochloride (TMB), hydrogen peroxide were obtained from Sigma Chemical Co. (St. Louis, USA.). A molecular weight standard of DNA was obtained from New England Biolabs (Beverly, MA) and a protein standard for electrophoresis from Sigma Chemical Co. (St. Louis, MO). Agarose, ampicillin, ethidium bromide, Trizma base, imidazole were purchased from

(34)

18

Sigma Chemical Co. (St. Louis, MO). Bacto-peptone, tryptone, yeast extract, and bacto-agar was purchased from Difco Laboratories (USA). All chemicals were of reagent grade. HiTrapFFfor purification of proteins fused with the His 6 residues and PD-10 desalting column were purchased from GE healthcare (Sweden). Quick Start Bradford Protein Assay Kit 2 was purchased from Bio-rad (USA).

1.2 Oligonucleotides

Oligonucleotides and codon optimized scFv gene were synthesized by Bioneer (Korea) (Fig. 9 and Fig. 10). They were synthesized with different restriction enzyme sites on the ends as primers according to the purpose of experiments, which are PCR amplication, construction of vector and recombination of gene.

1.3 Strains and plasmids

Escherichia coli Top10 [F- mcrA ∆(mrr-hsdRMS-mcrBC) φ80lacZ∆M15 ∆lacX74 recA1 araD139 ∆(ara-leu)7697 galU galK rpsL (StrR_{) endA1} nupG] (Invitrogen, CA, USA) was used for plasmid preparation for DNA manipulation. E. coli BL21(DE3) [F- ompT gal dcm lon hsdSB (rB-, mB-) λ(DE3 [lacI lacUB6-T7 gene 1 lnd1 sam7 nin5])] (Novagen, Germany) and E. coli C41(DE3) [F- ompT hsdSB (rB-, mB-) gal dcm (DE3)] (Lucigen, United

(35)

19

States) were used as host strains for the expression of target proteins. E. coli Origami (DE3) [ F-ompT hsdSB(rB- mB-) gal dcm lacY1 ahpC (DE3) gor522:: Tn10 trxB (KanR_{, Tet}R_{)] was used as host strains for expression of target} protein without signal sequence to periplasm.

Plasmids, pET19b were used as mother vectors which has the T7 promoter for expression of AFB1 scFv and maltose binding protein (MBP) fusion proteins. Aflatoxin B1 scFv genes was from plasmids of Won-Ki Min and Jae-Chan Park (Fig. 6). Fumonisin B1 and deoxynivalenol scFv genes were respectively from Jun-Bock Park (Fig. 7) and Gyu-Ho Choi (Fig. 8). MBP genes were from pMAL-p2X vector (New England Biolabs, USA) by polymerase chain reaction (PCR).

1.4 Recombinant DNA techniques

All of cloning steps were carried out according to the procedure of Sambrook et al (Sambrook et al., 1989). Mini-scale plasmid preparation was carried out with the High Pure Plasmid Isolation kit (Roche, Switzerland) and large-scale plasmid preparation was achieved using Plasmid Midi Kit (Qiagen, USA). Digestion of DNAs with restriction endonuclease, and dephosphorylation were achieved under the conditions recommended by the supplier, and the results of digestion were analyzed by agarose gel electrophoresis. This system was provided by Bio-Rad (USA). The isolation

(36)

20

of DNA fragments from agarose gel, solubilization of the gels and recovery of the DNA fragments were conducted by “High Pure PCR Product Purification Kit” from Roche (Switzerland) or “QIAquick Gel Extraction Kit” from Qiagen (USA). Ligation of the DNA fragments was carried out by “DNA Ligation Kit” from Takara (Japan).

1.4.1 Polymerase chain reaction (PCR)

All PCRs for amplification were performed with the GeneAmp 2400 (Applied Bioscience, USA). AccuPower PCR PreMix (Bioneer Co., Daejon, Korea), ready-to-use PCR reagent fully optimized for more accurate PCR amplification, was used in the PCR reaction. PCRs for cloning of genes were performed containing 10 pM each of forward and reverse primers, and plasmid DNA as a template. Reaction time and temperature were optimized according to each reaction condition. The amplified gene was confirmed by gel electrophoresis. Primers in this study are listed in Table 4.

1.4.2 Construction of expression plasmids

Expression plasmids, pET19b s.s malE Xa scFv H6, pET19b H6 scFv Xa malE, pET19b s.s malE Xa scFV op H6, pET19b pelB scFv op Xa malE H6, pET19b malE Xa scFV op H6, and pET19b H6 scFv op Xa

(37)

21

malE were constructed from pET19b, which purchased from Novagen (USA). The factors to be analyzed are codon optimization gene, direction of MBP fusion protein, existence of signal sequence, and plasmids expression hosts. The 6 histidine residues were fused to the N and C-terminal for efficient purification of proteins. The ligation of the expression vectors was performed using the DNA ligation kit according to manufacturer’s protocols.

1.5 DNA sequencing

DNA sequencing was performed by Mbiotech (Hanam, Korea). The results of DNA sequencing for selected clone candidates were compared each other, and then the selected DNAs were used in the subsequent experiments.

2. Expression of proteins

2.1 Transformation and expression of fusion proteins

Transformation of E. coli was carried out as described by Sambrook et al. (Sambrook et al., 1989). An E. coli strain Top10 was incoculated in 5 mL LB medium, and precultured at 37°C overnight. A 1% aliquot of the cultured cells was transferred into 50 mL of fresh LB medium and incubated with

(38)

22

shaking until OD at 600 nm reached 0.5. The pellet which had been harvested by centrifugation at 6,000 rpm for 5 min at 4°C was resuspended cautiously in 5 mL of ice-cold 100 mM CaCl2 solution and stored on ice for 30 min. The cell suspension was centrifuged (6,000 rpm for 5 min at 4°C) and the pellet was resuspended in 5 mL of 100 mM CaCl2 solution. Resuspended cells were aliquoted to 100 ㎕, mixed with ligated DNA, and kept on ice for 30 min. They were subjected to heat-shock at 42°C for 45 seconds. One mL of LB medium was added to the cells and incubated at 37°C for 1 hour with slow agitation. An appropriate volume of the transformed cells was spread on an LB agar plate with an ampicillin selection marker.

Equal amounts of scFv and maltose binding protein fusion plasmids were used for transformation of E. coli BL21(DE3) and C41(DE3). The transformed cells were spread on LB agar plates containing 50 µg/mL ampicillin (selection for plasmids).

E. coli BL21(DE3) and C41(DE3) colonies were picked from LB agar plates with ampicillin containing fresh transformed cells and cultured in 5 mL LB medium in the presence of ampicillin for about 12 hr at 37°C. Flask cultures were carried out with LB medium in 500 mL baffled flasks with a 100 mL working volume. Each flask was inoculated with 1.0 mL of the transformed

(39)

23

cell, pre-cultured in the log phase and was grown at 37°C in LB medium containing selective markers. Expression of scFv and MBP fusion proteins were induced by the addition of isopropyl-2-D-thio-galactopyranoside (IPTG) to final concentration of 0.2 mM.

2.2 SDS-PAGE

After induction, the induced cells were grown for about additional 4 h, were centrifuged at 10,000 rpm, resuspended in 100 mM sodium phosphate buffer (pH 7.4) and then were treated by sonication for cellular protein assay. The crushed cells were centrifuged at 12,000 rpm at 4°C for 15 min and the supernatant, taken as the soluble fraction, was recovered carefully. The pellet, taken as the insoluble fraction, was resuspended in an equivalent volume of the same phosphate buffer. Separation of proteins by molecular weight in SDS-PAGE, soluble and insoluble proteins were resuspended in sodium dodecyl sulfate (SDS) sample buffer and boiled at 100°C for 5 min. Heat-denatured fractions were analyzed by 10 ~ 15% SDS-polyacrylamide gel electrophoresis. Protein samples were electrophoresed on an SDS-containing discontinuous polyacrylamide gel electrophoresis unit using the Mini-Protein II system (Bio-Rad, USA). 12.5% (w/v) separating gel was prepared usually from 33.5% (w/v) acrylamide/ 0.3% (w/v) N, N’-methylenebisacrylamide stock solution in 0.38 M Tris-HCl (pH 9.1) and 0.1% (w/v) SDS. The 4%

(40)

24

(w/v) stacking gel was prepared from 30% (w/v) acrylamide/0.44% (w/v) N, N’-methylenebisacryl-amide stock solution in 0.125 M Tris-HCl (pH 6.8) and 0.1% (w/v) SDS. Both gels were polymerized with ammonium persulfate and TEMED. The running buffer was composed of 25 mM Tris base, 192 mM glycine, and 0.1% (w/v) SDS. Samples were mixed with equal volumes of 2X loading buffer [0.125 M Tris-Cl buffer (pH 6.8); 10% (v/v) β-mercaptoethanol; 4% (w/v) SDS; 20% (v/v) glycerol; a pinch of bromophenol blue] and boiled for 3 min before loading on the gel. Electrophoresis was carried out at 90 V for stacking and at 120 V for separating. When electrophoresis was finished, the gel was stained with commassie blue R-250 solution [0.2 % (w/v) Coomassie blue R-250; 50 % (w/v) methanol; 10 % (v/v) acetic acid] for 30 min with gentle shaking and destained with destaining solution [20 % (v/v) methanol; 10 % (v/v) acetic acid].

2.3 Fed-batch fermentation

Fed-batch culture was carried out in a 2.5 L jar fermentor (Kobiotech, Seoul, Korea) with a 1 L start working volume of a Riesenberg medium. The 100 ml seed culture was prepared in a 500 ml flask and grown in a shaking incubator at 37°C and 250 rpm for 12 hr, and main culture was carried out. To maintain the dissolved oxygen (DO) level, agitation speed and aeration

(41)

25

rate were in between 1200 rpm and 1 vvm of air supply, respectively. The pH was automatically controlled at 6.8 by the pH-stat strategy. To keep the cell growth and basal level of sugar after depletion of 20 g/L sugar initially added, 28% ammonia water and high concentration of carbon source feeding solutions (800 g/L glucose and magnesium sulfate heptahydrate 20 g/L) that were used. Feeding solutions were conversed to organic acids by the metabolic processes of cells. Expression of scFv and MBP fusion proteins was induced by the addition of IPTG when O.D. at 600 nm reached 95.

3. Purification and quantitative analysis of scFv

3.1 Purification

3.1.1 Affinity chromatography

The purification of the expressed protein was carried out by the Äcta prime system (Amersham Bioscience, Sweden) using the HisTrap FF column (GE healthcare, Sweden) used in His-tagged protein purification. This column was washed with distilled water. After equilibrating the column with binding buffer [20 mM sodium phosphate (pH 8.0), 500 mM NaCl], the sample resuspended in binding buffer [20 mM sodium phosphate (pH 8.0), 500 mM NaCl] was loaded. The column loaded with the sample was washed with binding buffer, and bound proteins were eluted with elution buffer [20 mM

(42)

26

sodium phosphate (pH 8.0), 500 mM NaCl, 500 mM immidazole] under constant level of immidazole concentration. Purified proteins were analyzed by SDS-PAGE (Fig. 11).

3.1.2 Desalting column

Desalting of purified protein was carried out using PD-10 desalting columns (GE healthcare, Sweden) by gravity force. The columns were washed with PBST buffer. After equilibrating the columns with PBST buffer, 2.5 mL of the protein samples was loaded to the columns. Bound proteins were eluted with 3.5 mL of PBST buffer (Fig. 11).

3.2 Quantitative analysis

3.2.1 Bradford assay

BSA (Bovine Serum Albumin) standard protein was prepared to determine standard curve for quantitative analysis of scFv and MBP fusion proteins. BSA with concentrations of 0, 2, 4, 5 µg/mL for the standard assay was used. scFv and MBP fusion protein samples were diluted with distilled water. 200 µl of Quick Start Bradford protein 1x dye reagent from Quick Start Bradford Assay Kit and 800 µl of protein were combined and vortexed. Combined samples were incubated at room temperature for 5 minutes, and

(43)

27

measured absorbance at 595 nm.

4. Immunological and physico-chemical analysis

4.1 Immunological analysis

4.1.1 Indirect ELISA

Antigen-binding affinity of scFv and MBP fusion proteins was examined by indirect ELISA (Enzyme linked immunosorbent assay) (Fig. 12). Antigen-binding activity was determined by increasing signal of scFv and MBP fusion proteins according to increasing concentration of antigen. Immune 96 Microwell Plate was coated with 0, 1, 5, 10, 50, 100, 1000, 10000 ppb of AFB1-BSA conjugate at room temperature overnight. The plate was washed by filling the wells with 200 µl of PBST buffer. After washing, plate was coated with 200 µl of 5% skim milk in 2 hours. The plate was washed with PBST buffer and coated with 100 µl of the prepared each of soluble scFv (0.01 g/L) and MBP fusion proteins and incubated at room temperature 2 hour. After washing, 100 µl of HisProbe-HRP was respectively added to each well and incubated 15 minutes. After washing, 100 µl of 5'-tetramethyl benzidine dihydrochloride (TMB) substrate solution was added and incubated in 30 minutes. 2 M sulfuric acid solution was added

(44)

28

to each well to stop color development. Absorbance of samples was measured with a microplate reader at 450 nm.

4.2 Physico-chemical analysis

4.2.1 Circular Dichroism Detector

Secondary structure of scFv and MBP fusion proteins in PBST buffer was analyzed by circular dichroism detector (Chirascan plus, Applied Photophysics, UK). Detector was High performance UV-Vis-IR avalanche photo-diode fluorescence monochro detector. Analyzing temperature was 25°C. Cell path length was 0.5 mm. Wavelength of CD was 190 ~ 250 nm to analyze secondary structure of proteins. PBST buffer was analyzed to determined baseline. Factor Xa protease treatment was conducted to obtain scFv without MBP to analyze secondary structure. Though, pure scFv itself was extremely unstable and remained as insoluble aggregate (Fig. 30). Thus, MBP and scFv-MBP fusion protein were analyzed. Absorbance difference of MBP and MBO-scFv was analyzed.

(45)

29

5. Application of expression system to other types of scFv

5.1 Fumonisin B

1

scFv

5.1.1 Expression of fumonisin B

1

scFv and MBP fusion

protein in E. coli C41(DE3) strain

E. coli BL21(DE3) and C41(DE3) carrying fumonisin B1 scFv and MBP fusion protein plamids were inoculated in LB medium baffled flask and grown at 37°C, 250 rpm. Expression of proteins was induced by the addition of isopropyl-2-D-thio-galactopyranoside (IPTG) to final concentration of 0.2 mM. Expression was analyzed by SDS-PAGE.

5.2 Deoxynivalenol scFv

5.2.1 Expression of deoxynivalenol scFv and MBP

fusion protein in E. coli C41(DE3) strain

E. coli BL21(DE3) and C41(DE3) carrying deoxynivalenol scFv and MBP fusion protein plamids were inoculated in LB medium baffled flask and grown at 37°C, 250 rpm. Expression of proteins was induced by the addition of isopropyl-2-D-thio-galactopyranoside (IPTG) to final concentration of 0.2 mM. Expression was analyzed by SDS-PAGE.

(46)

30

Table 4. Sequence of the primers used in this research

Primer name Primer sequences

(5’-3’)

MalE-BspHI-F AAG CTT TC ATG AAA ATA AAA ACA GGT GCA CGC MalE-NdeI-R AAT CGC CAT ATG CCT TCC CTC GAT CCC GAG GTT pelBscFv AFB-BspHI-F AAG CTT TC ATG AAA TAC CTG CTG CCG ACC

pelBscFv AFB-NdeI AAT CGC CAT ATG CCT TCC CTC GAT ACC TAG GAC GAG TTT GGT TCC TC

scFv AFB H6-BamH1-R AAT CGC GGA TCC TTA GTG GTG GTG GTG GTG GTG C His6 scFv AFB-Nde1-F AAT CGC CAT ATG CAC CAC CAC CAC CAC CAC ATG GAG GTG AAG

CTG CAG

His6 scFv AFB Xa-xho1-R AAT CGC CTC GAG CCT TCC CTC GAT ACC TAG GAC GAG TTT GGT TCC TC

malE-xhoI-F2 AAT CGC CTC GAG AAA ATC GAA GAA GGT AAA CTG GTA ATC malE-BamHI-R2 AAT CGC GGA TCC TTA CCC GAG GTT GTT GTT ATT GTT ATT G scFv AFB1 H6-Nde1-IF-F TCGAGGGAAGGCATATGGAGGTGAAGCTGCAGGAGTCTG scFv AFB1 H6-BamH1-IF-R GTTAGCAGCCGGATCCTTAGTGGTGGTGGTGGTGGTGC

(47)

31

scFv op. H6-Nde1-IF-F TC GAG GGA AGG CAT ATG GAA GTG AAA CTG CAG GAA AGC G scFv op. H6-BamH1-IF-R G TTA GCA GCC GGA TCC TCA GTG GTG GTG GTG GTG GTG C

pelB scFv op. - Nde1 – F AAT CGC CAT ATG AAA TAC CTG CTG CCG ACC GC pelB scFv op. Xa - xho1 – R AAT CGC CTC GAG CCT TCC CTC GAT GCC CAG CAC CAG TTT GGT G

malE H6-BamHI-R2 AAT CGC GGA TCC TTA GTG GTG GTG GTG GTG GTG CCC GAG GTT GTT GTT ATT GTT ATT G

malE Xa – BspH1-F AAG CTT TC ATG AAA ATC GAA GAA GGT AAA CTG ATC H6 scFv op Xa – Nde1 – F AATCGCCATATGCACCACCACCACCACCAC ATG GAA GTG AAA CTG

CAG GAA AGC

H6 scFv op Xa – Xho1 – R AAT CGC CTC GAG CCT TCC CTC GAT GCC CAG CAC CAG TTT GGT G pelB scFv op. Xa – BspH1 – F AAG CTT TC ATG AAA TAC CTG CTG CCG ACC GC

pelB scFv op. Xa – Nde1 – R AAT CGC CAT ATG CCT TCC CTC GAT GCC CAG CAC CAG TTT GGT G malE H6-Nde1-F2 AAT CGC CAT ATG AAA ATC GAA GAA GGT AAA CTG GTA ATC malE H6-XhoI-R2 AAT CGC CAT ATG AAA ATC GAA GAA GGT AAA CTG GTA ATC FB1 scFv - Nde1 – F AAT CGC CAT ATG GAT GTA GTC ATG ACC CAG TCT CC FB1 scFv - BamH1 – R AAT CGC GGA TCC TCA GTG GTG GTG GTG GTG GTG

DON scFv - Nde1 – F AAT CGC CAT ATG CAG GTG AAG CTG CAG CAG TCT G DON scFv - BamH1 – R AAT CGC GGA TCC TCA GTG GTG GTG GTG GTG GTG

(48)

32 MKIKTGARILALSALTTMMFSASALAKIEEGKLVIWINGDKGYNGLA EVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFG GYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSL IYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNA DTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKG QPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPL GAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRT AVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGIEGRHMEV KLQESGGGLVKPGGSLKLSCAASGFTFSTYAMSWVRQTPEKRLEWV ATISSGGTYTYSPDSVKGRFTISRDNAKNTLYLQMSSLRSEDTAMYY CASHGLLWSFAYWGQGTTVTVSSGGGGSGGGGSGGGGSQAVVTQE SALTTSPGETVTLTCRSSTGAVTTSNSANWVQEKPDHLFTGLIGGTN NRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHLVFG GGTKLVLGLEHHHHHH

(49)

33 MDVVMTQSPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPG QSPKRLIYLVSKLDSGFPDRFTGSGSGTDFTLKISRVEAEDLGVYYCW QGIHFPRTFGGGTKLEMGGGGSGGGGSGGGGSEVQLQQSGAELVKP GASVKLSCKTSGYTFTSYWIQWVKQRPGQGLGWIGEIFPGTGTTYY NEKFKGKATLTIDTSSSTVYMQLSSLTSEDSAVYFCASRRFAYWGQG TTVTVSSLEHHHHHH

Fig. 7. Amino acid sequence of fumonisin B1 scFv with 6 histidine tag

MQVKLQQSGTEVVKPGASVKLSCKASGYIFTSYDIDWVRQTPEQGL EWIGWIFPGEGSTEYNEKFKGRATLSVDKSSSTAYMELTRLTSEDSA VYFCARGDYYRRYFDLWGQGTTVTVSSGGGGSGGGGSGGGGSQAV VTQESALTTSPGGTVILTCRSSTGAVTTSNYANWVQEKPDHLFTGLI GGTSNRAPGVPVRFSGSLIGDKAALTITGAQTEDDAMYFCALWYST HFVFGGGTKVTVLGLEHHHHHH

(50)

34

(51)

35

(52)

36

Fig. 11. Purification process

Cell lysis by sonication

Collection of soluble fraction

Affinity chromatography

(Histag purification)

Desalting

(Buffer change to PBST buffer

for ELISA)

(53)

37

(54)

38

III. RESULTS AND DISCUSSIONS

1. Plasmids and strains

1.1 Construction of expression plasmids and strains

To obtain soluble and functional scFv in vivo, maltose binding protein which is expressed in high solubility in E. coli was fused with AFB1 scFv. Direction of MBP fusion to scFv such as N or C-terminal influences expression of proteins. Both direction fusion protein expression plasmids were constructed with scFv and codon optimized scFv gene. Factor Xa protease site was inserted between scFv and MBP gene. For purification, 6 hisitidine tag was combined into the expression plasmids. pET19b s.s. malE Xa scFv H6 and pET19b H6 scFv Xa malE plasmids were constructed for non-codon optimized gene. pET19b s.s malE Xa scFv op H6, pET19b pelB scFv op Xa malE H6, pET19 malE Xa scFv op H6, and pET19 H6 scFv op Xa malE were constructed for codon optimized scFv gene. All plasmids usded and constructec in this research are summarized from fig. 13 to 16 and Table 5.

(55)

39

2. Expression of proteins

2.1 scFv and MBP fusion protein expression

In order to express soluble and active scFv and elevate the expression level of scFv, maltose binding protein was fused. pET19 s.s malE Xa scFv H6 and pET19 H6 scFv Xa malE were expressed in E. coli BL21(DE3). Control strains are E. coli BL21(DE3) carrying scFv gene without MBP fusion protein.

Maltose binding protein fusion slightly elevated the expression level in E. coli BL21(DE3) strains (Fig. 17 and Fig. 18). However, majority of expressed scFv and MBP fusion protein was insoluble. Experimental results gave no positive effect on sloluble expression.

2.2 Codon optimized and non-codon optimized scFv gene

expression in diverse E. coli host strains.

Codon optimized scFv gene to optimized translation in E. coli was fused with maltose binding protein in N and C-terminal. Periplasmic targeting signal sequence of pelB or maltose binding protein itself was inserted in express vectors.

Some of MBP fusion optimized scFv expressed in BL21(DE3), though expression pattern was insoluble (Fig. 19 to 20). N-terminal MBP fusion

(56)

40

scFv with signal sequence in C41(DE3) host expressed in soluble form. (Fig. 22) Fusion protein without signal sequence was expressed in Origami (DE3) strains (Fig. 23). scFv and MBP protein expression in Origami (DE3) strains was no positive effect (Fig 24).

To verify the direction of fusion protein and compare with these result of experiments with non-codon optimized scFv gene, non-codon optimized gene was fused with maltose binding protein in N and C-terminal of scFv. Like the preceding, N-terminal MBP fusion scFv with signal sequence in C41(DE3) was solely expressed soluble form of proteins (Fig. 21). These results suggested that expression system of scFv and MBP fusion in E. coli C41(DE3) strains enhanced soluble expression level of scFv proteins.

2.3 Fed-batch fermentation

Fed-batch fermentations were conducted with the recombinant strains which produced the largest amount of solube scFv and MBP fusion proteins – C41(DE3) carrying pET19 s.s malE Xa scFv H6. After initial glucose concentration of 20 g/L was completely exhausted, the operation mode was switched to the pH-stat fed-batch by using feeding solution. When O.D. at 600 nm reached 80 ~ 100, IPTG (0.2 mM) were added.

(57)

41

scFv H6 resulted in 49.275 g/L dry cell weight, 810 mg/L scFv and MBP fusion protein concentration (Fig. 25). Total glucose consuption was 310 g. Soluble fraction of scFv and MBP fusion protein produced by fed-batch fermentation had purification of affinity chromatography. Fed-batch profile is shown (Fig. 25).

3. Purification and quantitative analysis of scFv

For the efficient purification of the scFv and MBP fusion proteins, 6 His residues were fused. The HisTrap FF column (GE healthcare, Sweden) specific for His 6 residues was used. FPLC was operated to purify histagged scFv and MBP proteins. SDS-PAGE analysis indicated that the target antibody was purified and detected at the size of molecular weight of 27 kDa. (Fig. 26) These results indicate that construction of in vivo scFv and MBP fusion protein production system.

To quantify the scFv and MBP fusion proteins, standard curve using BSA solution was prepared. Using standard curve, scFv and MBP fusion protein was quantified to be used for immunological and physic-chemical analysis.

(58)

42

4. Immunological and physic-chemical analysis

4.1 Indirect ELISA

To analyze binding activity of AFB1 scFv and MBP fusion protein to aflatoixn B1, indirect ELISA was carried out. The detection limit of aflatoxin B1 was 50 ppb (ng/ml). Antigen-binding activity was confirmed by increasing signal of scFv and MBP fusion proteins according to increasing concentration of antigen (Fig. 27 to Fig. 29).

4.2 Circular dichroism

To analyze secondary structure of scFv, circular cichroism was carried out. Alpha helix rich protein has round lowest point at 208 and 222 nm. Beta sheet rich protein has point at 218 nm. Secondary structure of scFv is beta sheet rich protein. MBP describe as most secondary structure is involved in alpha-helical structure. Signal of scFv and MBP fusion protein minus signal of MBP protein is scFv signal. These subtraction result present that scFv has beta sheet rich secondary structure and folded properly (Fig. 31).

(59)

43

5. Application of expression system to other types of scFv

5.1 Fumonisin B

1

scFv

Above-described, expression system of scFv and MBP fusion in E. coli C41(DE3) strains enhanced soluble expression level of scFv proteins. There are two types of scFv according to the light chain such as lambda and kappa. For broad applications of this expression s system, other types of scFv were examined. Aflatoxin B1 scFv is lambda type. Fumonisin B1 scFv from Jun-Bock Park is kappa type scFv. After analyzing the expression of scFv and MBP fusion protein using this expression system by SDS-PAGE, there was positive effect on soluble expression (Fig. 32).

5.2 Deoxynivalenol scFv

Deoxynivalenol scFv from Gyu-Ho Choi is lambda type like aflatoxin B1 scFv. . After analyzing the expression of scFv and MBP fusion protein using this expression system by SDS-PAGE, there was positive effect on soluble expression. Gathering up these results from aflatoxin B1 scFv, fumonisin B1 scFv, and deoxynivalenol scFv, it propose that expression system of scFv and MBP fusion protein expression in E. coli C41(DE3) has certain effect on producing soluble scFv (Fig. 33).

(60)

44

Fig. 13. Genetic map of non-codon optimized scFv gene for aflatoxin B1

pET19b. 7619 bp lacI Amp . T7 promoter factor Xa Ori . . pET19b H6 scFv Xa malE His tag

Maltose binding protein without signal sequence

scFv pET19b. 7619 bp lacI Amp His tag T7 promoter factor Xa Ori . . pET19b s.s malE Xa scFv H6 Maltose binding protein

with signal sequence scFv

(61)

45

Fig. 14. Genetic map of codon optimized scFv gene with signal sequence for aflatoxin B1 pET19b. 7619 bp lacI Amp His tag T7 promoter factor Xa Ori . . pET19b s.s malE Xa scFv op H6 Maltose binding protein

with signal sequence

optimized scFv pET19b. 7619 bp lacI Amp . His tag T7 promoter factor Xa Ori . . pET19b pelB scFv op Xa malE H6 Optimized scFv

pelB signal sequence Maltose binding protein

(62)

46

Fig. 15. Genetic map of codon optimized scFv gene without signal sequence for aflatoxin B1 pET19b. 7619 bp lacI Amp . T7 promoter factor Xa Ori . . pET19b H6 scFv op Xa malE His tag

Maltose binding protein without signal sequence

Optimized scFv pET19b. 7619 bp lacI Amp His tag T7 promoter factor Xa Ori . . pET19b malE Xa scFv op H6 Maltose binding protein

without signal sequence optimized scFv

(63)

47

Fig. 16. Genetic map of fumonisin B1 and deoxynivalenol scFv gene with signal sequence for aflatoxin B1

pET19b. 7619 bp lacI Amp His tag T7 promoter factor Xa Ori . . Maltose binding protein

with signal sequence DON scFv pET19b s.s malE Xa DON scFv H6 pET19b. 7619 bp lacI Amp His tag T7 promoter factor Xa Ori . . pET19b s.s malE Xa FB1 scFv H6 Maltose binding protein

with signal sequence FB1 scFv

(64)

48

Table 5. Constructed strains used in this research

Strain Plasmids E. coli BL21(DE3) pET19b s.s malE Xa scFv H6 E. coli C41(DE3) E. coli BL21(DE3) pET19b H6 scFv Xa malE E. coli C41(DE3) E. coli Origami(DE3) E. coli BL21(DE3) pET19b s.s malE Xa scFv op H6 E. coli C41(DE3) E. coli BL21(DE3)

pET19b pelB scFv op Xa malE H6

E. coli C41(DE3) E. coli BL21(DE3) pET19b malE Xa scFv op H6 E. coli C41(DE3) E. coli Origami(DE3) E. coli BL21(DE3) pET19b H6 scFv op Xa malE E. coli C41(DE3) E. coli Origami(DE3)

(65)

49

Fig. 17. Expression of non-MBP fusion scFv in BL21(DE3) at 37°C and 0.2 mM IPTG. L T S I T S I 30 kDa 40 kDa 50 kDa 25 kDa BL21(DE3)/pET26 pelB scFv H6 AFB1 scFv : 26.5 kDa MBP: 42 kDa

scFv-MBP fusion protein: 68.5 kDa

L : Protein Ladder

T: Total (Before Induction)

T: Total (After Induction)

S : Soluble (After Induction)

(66)

50

Fig. 18. Expression of MBP fusion scFv in BL21(DE3) at 37°C and 0.2 mM IPTG.

AFB1 scFv : 26.5 kDa MBP: 42 kDa

I : Insoluble (After Induction)

BL21(DE3) pET19b s.s malE Xa scFv H6

40 kDa 50 kDa 60 kDa 70 kDa T T S I L 30 kDa 40 kDa 50 kDa 60 kDa 70 kDa L T L T S I BL21(DE3)/pET19b H6 scFv Xa malE

(67)

51

Fig. 19. Expression of MBP fusion codon optimized scFv with signal sequence in BL21(DE3) at 37°C and 0.2 mM IPTG.

30 kDa 40 kDa 50 kDa 60 kDa 70 kDa T T S I L

BL21(DE3)/ pET19b s.s malE Xa scFv op. H6

30 kDa 40 kDa 50 kDa 60 kDa 70 kDa

BL21(DE3)/pET19b pelB scFv op. Xa malE H6

L T L T S I

(68)

52

Fig. 20. Expression of MBP fusion codon optimized scFv without signal sequence in BL21(DE3) at 37°C and 0.2 mM IPTG.

30 kDa 40 kDa 50 kDa 60 kDa 70 kDa L T L T S I BL21(DE3)/pET19b malE Xa scFv op. H6

30 kDa 40 kDa 50 kDa 60 kDa 70 kDa

BL21(DE3)/pET19b H6 scFv op. Xa malE

T T S I L

(69)

53

Fig. 21. Expression of MBP fusion scFv in C41(DE3) at 37°C and 0.2 mM IPTG. C41(DE3)/pET19 H6 scFv Xa malE 30 kDa 40 kDa 50 kDa 60 kDa 70 kDa 25 kDa L T T S L I C41(DE3)/pET19 s.s malE Xa scFv H6 30 kDa 40 kDa 50 kDa 60 kDa 70 kDa 25 kDa L T T S I AFB1 scFv : 26.5 kDa MBP: 42 kDa

(70)

54

Fig. 22. Expression of MBP fusion codon optimized scFv with signal sequence in C41(DE3) at 37°C and 0.2 mM IPTG.

C41(DE3)/pET19b s.s malE Xa scFv op. H6 L T L T S I 40 kDa 50 kDa 60 kDa 70 kDa 30 kDa 40 kDa 50 kDa 60 kDa 70 kDa

C41(DE3)/pET19b pelB scFv op. Xa malE H6 L T T L S I

(71)

55

Fig. 23. Expression of MBP fusion codon optimized scFv without signal sequence in C41(DE3) at 37°C and 0.2 mM IPTG.

C41(DE3)/pET19b malE Xa scFv H6 30 kDa 40 kDa 50 kDa 60 kDa 70 kDa L T T S I 30 kDa 40 kDa 50 kDa 60 kDa 70 kDa C41(DE3)/pET19b H6 scFv op Xa malE T T S I L AFB1 scFv : 26.5 kDa MBP: 42 kDa

(72)

56

Fig. 24. Expression of MBP fusion codon optimized scFv without signal sequence in Origami (DE3) at 37°C and 0.2 mM IPTG

Origami (DE3)/ pET19b malE Xa scFv op H6

30 kDa 40 kDa 50 kDa 60 kDa 70 kDa T T S I L L T L T S I

Origami (DE3)/pET19 H6 scFv op Xa malE

30 kDa 40 kDa 50 kDa 60 kDa 70 kDa L : Protein Ladder

I : Insoluble (After Induction)

(73)

57

Fig 25. Profile of fed-batch fermentation Time (h) 0 5 10 15 20 25 30 D C W , G lu c o s e ( g /L ) 0 10 20 30 40 50 60 sc F v ( m g /L ) 0 200 400 600 800 1000 DCW Glucose scFv

(74)

58

Fig 26. Purification of scFv and MBP fuison protein 30 kDa 40 kDa 50 kDa 60 kDa 70 kDa L E1 E2 E3 E4 E5 E6 E7 L [His tag purification]

L : Protein Ladder F : Flow-through fraction E : Elution fraction 30 kDa 40 kDa 50 kDa 60 kDa 70 kDa [Desalting column] AFB1 scFv : 26.5 kDa MBP: 42 kDa

(75)

59

Fig 27. Antigen (aflatoxin B1) binding activity of non-codon optimized scFv and MBP fuison protein by indirect ELISA

MBP-scFv ELISA Con. of aflatoxin (ppb) 1 5 10 50 100 1000 10000 A45 0 0.000 0.500 1.000 1.500 2.000 scFv

(76)

60

Fig 28. Antigen (aflatoxin B1) binding activity of codon optimized scFv and MBP fuison protein by indirect ELISA

MBP-scFv op. ELISA Con. of aflatoxin (ppb) 1 5 10 50 100 1000 10000 A45 0 0.000 0.500 1.000 1.500 2.000 scFv-op

(77)

61

Fig 29. Color reaction of substrate to measure antigen binding activity by indirect ELISA

Aflatoxin B1-BSA concentration 0 ppb ~ 104 ppb

s.s malE Xa scFv H6 0.01 g/L

s.s malE Xa scFv op H6 0.01 g/L

(78)

62

Fig 30 Factor Xa protease treatment to take scFv itself

T: Total (0 hour protease treatment )

T: Total (1 hour protease treatment )

S : Soluble (1 hour protease treatment )

I : Insoluble (1 hour protease treatment )

scFv-MBP fusion protein: 68.5 kDa Factor Xa protease: 43 kDa

L T L T S I 30 kDa 40 kDa 50 kDa 60 kDa 70 kDa 25 kDa

(79)

63 scFv Wavelength (nm) 180 200 220 240 260 280 [Q ]MR W ( d eg c m 2 d m o l -1 ) -4e+6 -3e+6 -2e+6 -1e+6 0 1e+6 2e+6 scFv

(80)

64

Fig. 32. Expression of MBP fusion scFv of fumonisin B1 in C41(DE3) at 37°C and 0.2 mM IPTG. T T S I T T S I L BL21(DE3) MBP - FB1 scFv C41(DE3) MBP - FB1 scFv 30 kDa 40 kDa 50 kDa 60 kDa 70 kDa 25 kDa FB1 scFv : 27 kDa MBP: 42 kDa

scFv-MBP fusion protein: 69 kDa

(81)

65

Fig. 33. Expression of MBP fusion scFv of deoxynivalenol in C41(DE3) at 37°C and 0.2 mM IPTG. T T S I L L T L T S I BL21(DE3) MBP - FB1 scFv _{MBP - FB1 scFv}C41(DE3) 30 kDa 40 kDa 50 kDa 60 kDa 70 kDa 25 kDa DON scFv : 27 kDa MBP: 42 kDa

scFv-MBP fusion protein: 69 kDa

(82)

66

V. CONCLUSIONS

A robust expression system of E. coli C41(DE3) carrying the scFv-MBP fusion gene were developed to produce soluble and active scFv of aflatoxin B1. The results obtained in this research can be summarized as follows;

1) An expression system for aflatoxin B1 by fusing the scFv with MBP in E. coli C41(DE3) was developed for production of soluble scFv.

2) The expression system can be applied to produce other types of scFv in soluble form such as fumonisin B1 and deoxynivalenol. 3) The E. coli C41(DE3) strain was able to produce the maximum

scFv-MBP fusion protein level of 810 mg/L in fed-batch fermentation. Aflatoxin B1 binding activity of scFv and MBP fusion protein was confirmed. The secondary structure of scFv was determined to be the beta sheet by circular dichroism.

(83)

67

V. REFERENCES

Alefounder PR, Ferguson SJ (1980) The location of dissimilatory nitrite reductase and the control of dissimilatory nitrate reductase by oxygen in Paracoccus denitrificans. Biochemical Journal 192: 231-240

Ahmad ZA, Yeap SK, Ali AM, Ho WY, Alitheen BNM, Hamid M (2012) scFv antibody: Principles and clinical application. Clinical and

Developmental Immunology 2012: 1-15

Baneyx F, Mujacic M (2004) Recombinant protein folding and misfolding in Escherichia coli. Nature Biotechnology 22: 1399-1408

Bessette PH, Aslund F, Beckwith J, Georgiou G (1999) Efﬁcient folding of proteins with multiple disulﬁde bonds in the Escherichia coli cytoplasm. Procedings of the National Academy Sciences 96:13703–13708.

Chen G, Dubrawsky I, Mendez P, Georgiou G, Iverson BL (1999) In vitro scanning saturation mutagenesis of all the specificity determining residues in an antibody binding site. Protein Engineering 12: 349-356

Cho YJ, Lee DH, Kim DO, Min WK, Bong KT, Lee GG, Seo JH (2005) Production of monoclonal antibody against Ochratoxin A and its application to immunochromatographic assay. Journal of Agriculture and Food

Production of functional single-chain variable fragment specific for food-born mycotoxin, aflatoxin B1 in engineered Escherichia coli

Production of functional single-chain variable fragment

specific for food-born mycotoxin, aflatoxin B

in engineered Escherichia coli

재조합

대장균에서

곰팡이

독소 아플라톡신

특이적인

재조합

항체의 생산

By

Hyo-Ran Lee

School of Agricultural Biotechnology

Seoul National University

Production of functional single chain variable fragment

specific for food-born mycotoxin, aflatoxin B

in engineered Escherichia coli

Advisor: Professor Jin-Ho Seo

Submitted in Partial Fulfillment of the Requirements

for the Degree of Master of Science

By

Hyo-Ran Lee

School of Agricultural Biotechnology

Seoul National University

農學碩士學位論文

Production of functional single chain variable fragment

specific for food-born mycotoxin, aflatoxin B

in engineered Escherichia coli

재조합

대장균에서

곰팡이

독소 아플라톡신

특이적인

재조합

항체의 생산

ABSTRACT

CONTENTS

LIST OF TABLES

LIST OF FIGURES

I. INTRODUCTION

1. A

B

2. Detection methods of AFB

3. Single-chain variable region fragment antibody (scFv)

4. Codon optimization

5. Affinity tags

6. E. coli strains for protein expression

7. Circular Dichroism

7.

O

II. MATERIALS AND METHODS

1. Plasmids and strains

1.1 Enzymes and reagents

1.2

Oligonucleotides

1.3 Strains and plasmids

1.4 Recombinant DNA techniques

1.4.1 Polymerase chain reaction (PCR)

1.4.2 Construction of expression plasmids

1.5 DNA sequencing

2. Expression of proteins

2.1 Transformation and expression of fusion proteins

2.2 SDS-PAGE

2.3 Fed-batch fermentation

3. Purification and quantitative analysis of scFv

3.1 Purification

3.1.1 Affinity chromatography

3.1.2 Desalting column

3.2 Quantitative analysis

3.2.1 Bradford assay

4. Immunological and physico-chemical analysis

4.1 Immunological analysis

4.1.1 Indirect ELISA

4.2 Physico-chemical analysis

4.2.1 Circular Dichroism Detector

5. Application of expression system to other types of scFv

5.1 Fumonisin B

scFv

5.1.1 Expression of fumonisin B