Construction of expression plasmids and strains

III. RESULTS AND DISSCUSSIONS

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.

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

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.

The pH-stat fed-batch fermentation of BL21 (DE3) /pET19b s.s malE Xa

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.

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).

5. Application of expression system to other types of scFv 5.1 Fumonisin B

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).

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

pET19b.

Fig. 14. Genetic map of codon optimized scFv gene with signal sequence for aflatoxin B1

Fig. 15. Genetic map of codon optimized scFv gene without signal sequence for aflatoxin B1

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

pET19b.

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)

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

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) I : Insoluble (After Induction)

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

scFv-MBP fusion protein: 68.5 kDa

L : Protein Ladder

T : Total (Before Induction) T: Total (After Induction) S : Soluble (After Induction) I : Insoluble (After Induction)

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

40 kDa

BL21(DE3)/pET19b H6 scFv Xa malE

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

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

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) I : Insoluble (After Induction)

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

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

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) I : Insoluble (After Induction)

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

C41(DE3)/pET19 s.s malE Xa scFv H6

30 kDa

scFv-MBP fusion protein: 68.5 kDa

L : Protein Ladder

T : Total (Before Induction) T: Total (After Induction) S : Soluble (After Induction) I : Insoluble (After Induction)

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

C41(DE3)/pET19b pelB scFv op. Xa malE H6

L T

L

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) I : Insoluble (After Induction)

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

C41(DE3)/pET19b H6 scFv op Xa malE

T

T S I

L

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) I : Insoluble (After Induction)

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

Origami (DE3)/pET19 H6 scFv op Xa malE

30 kDa

scFv-MBP fusion protein: 68.5 kDa

Fig 25. Profile of fed-batch fermentation Time (h)

Fig 26. Purification of scFv and MBP fuison protein

30 kDa

scFv-MBP fusion protein: 68.5 kDa

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

A450

0.000 0.500 1.000 1.500 2.000

scFv

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

A450

0.000 0.500 1.000 1.500 2.000

scFv-op

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

Fig 30 Factor Xa protease treatment to take scFv itself

L : Protein Ladder

T : Total (0 hour protease treatment ) T: Total (1 hour protease treatment ) S : Soluble (1 hour protease treatment ) I : Insoluble (1 hour protease treatment )

AFB1 scFv : 26.5 kDa MBP: 42 kDa

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

L T L

T S

30 kDa 40 kDa 50 kDa 60 kDa 70 kDa

25 kDa

63 scFv

Wavelength (nm)

180 200 220 240 260 280

[Q]MRW (deg cm2 dmol-1 )

-4e+6 -3e+6 -2e+6 -1e+6

0 1e+6 2e+6

scFv

Fig 31. Secondary structure analysis of scFv by circular dichroism

Fig. 32. Expression of MBP fusion scFv of fumonisin B1 in C41(DE3) at 37°C and 0.2 mM IPTG.

scFv-MBP fusion protein: 69 kDa

L : Protein Ladder

T : Total (Before Induction) T: Total (After Induction) S : Soluble (After Induction) I : Insoluble (After Induction)

Fig. 33. Expression of MBP fusion scFv of deoxynivalenol in C41(DE3) at 37°C and 0.2 mM IPTG.

scFv-MBP fusion protein: 69 kDa

L : Protein Ladder

T : Total (Before Induction) T: Total (After Induction) S : Soluble (After Induction) I : Insoluble (After Induction)

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.

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국 문 초 록

국제기구인 International Agency of Research on Cancer (IARC)에서 지정한 독성이 강한 곰팡이 독소 aflatoxin B1 은 Aspergillus 속에 감염된 작물에서 발견되며 독소에 의한 질병 유발을 방지하기 위해 이를 검출하는 것이 중요하다. Aflatoxin

B1을 검출에 있어서 최근 많은 검체를 짧은 시간 내에 분석할 수 있는 면역학적 방법이 널리 연구되고 있으며, 한 방법으로 대장균 내에서 재조합 항체인 single chain variable fragment (scFv)를 생산하는 연구가 보고되었다. 선행연구에 의해

aflatoxin B1 의 scFv 가 monoclonal antibody 로부터 생산되었으나 이는 불용성 상태로 발현이 되었다. 뒤를 이은 선행연구에 의하여 대장균 내에서 활성을 갖는 scFv 를 생산하기 위하여 샤페론을 도입하고 대장균의 세포질내로 분비하도록 하여 활성형의 scFv 를 생산하였다. 하지만 발현 시에 활성형으로 발현되는 정도가 미약하여 이를 개선할 필요가 있었다.

본 연구에서는 새로운 발현 시스템을 목적으로 하여 첫째, 코돈 최적화를 통하여 대장균 내에서 단백질 번역과정이 수월하게 일어나도록 하였다. 하지만 코돈 최적화는 scFv 의 발현과정에 생기는 문제점을 해결할 수 없었다. 둘째,

scFv 를 가용성이 높은 단백질인 maltose binding protein (MBP)과 fusion 을 시도하였다. scFv 의 N 말단과 C 말단에 MBP 를 fusion 하였으며, signal sequence 의 유무에 관한 영향도 측정했다. 대장균 BL21(DE3) 균주에서 발현시켜 본 결과 발현양의 증가는 미약하게 나타나지만 불용성의 단백질로 발현되는 것을

문서에서 Production of functional single-chain variable fragment specific for food-born mycotoxin, aflatoxin B1 in engineered Escherichia coli (페이지 54-0)