Display of heterologous gene products on the Escherichia coli cell surface as fusion proteins with flagellin

JOURNAL OF FERMENTATION AND BIOENGINEERING

Vol. 86, No. 5, 500-503. 1998

Display of Heterologous Gene Products on the Escherichia coli Cell Surface as Fusion Proteins with Flagellin SATOSHI

EZAKI,’

MANAE

TSUKIO,*

MASAHIRO

TAKAGI,*

AND

TADAYUKI

IMANAKA’*

Department of Synthetic Chemistry and Biological Chemtitry, Graduate School of Engineering, Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto 606-8501’ and Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamada-oka, Suita, Osaka 565-0871,2 Japan Received 6 May 1998/Accepted 3 1 July 1998 We have developed a system for expressing and displaying recombinant proteins, on the surface of Escherichia coli cells using the flagellin gene (hag). In order to produce fusion proteins, DNA fragments encoding a small peptide with 15 amino acid residues (P15), the constant region of the antibody L chain (region C), a single chain Fv (sFv) of an anti-porphyrin antibody, green fluorescent protein (GFP) and a bacterial alkaline phosphatase (BAP), respectively were inserted into the dispensable D3 domain encoded by hag. Each fusion gene was expressed in E. coli YK4516, a strain that lacks hag. The peptide linker P15 and the region C were efficiently expressed and displayed in the flagellar fraction as fusion proteins. Although with lower efficiency, sFv, BAP and GFP could also be expressed in the flagellar fraction. Therefore, this system is useful for epitope analysis and with some improvements, the method could be useful for the expression of heterologous proteins on the ceil surface. [Key words:

flagellin, cell surface display, Escherichiu coli, secretion, fusion protein] ray diffraction

(16). Flagellin has three domains, Dl and D2 are needed for the formation of the functional flagellar filament, D3 does not take part in the formation of flagella (17). Therefore, in this study, we inserted the gene of an sFv (single chain Fv) (18) from an antibody, as well as some enzymes and proteins, into the flagellin gene (hag) at the position encoding the dispensable D3 domain and attempted to express flagellin fusion proteins. To prepare the flagellar fraction, cultures of E. coli were incubated at 65°C for 15 min to separate the flagella from cells. After centrifugation at 5000 xg for 5 min at room temperature, the supernatant was transferred to new tubes and 15 ml of cold acetone was added. After cooling at -20°C for 1 h, the protein precipitate was recovered by centrifugation at 10,000 x g for 10min at 4°C. The precipitates were dried and resuspended in SDS-sample buffer and applied for SDS-polyacrylamide gel electrophoresis. We could not detect any antibody L chain in the flagellar fraction of cells harboring the L chain gene alone by Western blot analysis, indicating that no cross contamination between flagellar fraction and intracellular fraction had occurred (data not shown). To investigate the possibility of the expression of fusion proteins using flagella, we prepared three types of chimeric flagellin (Fig. IA). The complete flagellin gene was inserted into the pTV119N (Takara Shuzo Co., Kyoto) to yield pTF1. The pTF2 plasmid series had a complete deletion of hag domain 3. The pTF3 plasmid series retained 30 bp of domain 3 at its boundaries with domain 1 and 2. The pTF4 series harbored restriction enzyme sites at the center of the complete domain 3 to enable cloning of the various insert genes. A 15-amino acid peptide linker (P15, (Gly, Serb) (19) was inserted into pTF2, pTF3 and pTF4, respectively. The resultant plasmids, pTF2K, pTF3K and pTF4K, were used to transform E. coli YK4516, which lacks the flagellin gene. Figure 1B shows SDS-PAGE of various flagellin-linker fusion proteins in the flagellar fraction. The amount of

Several systems for displaying small heterologous peptides on the surface of Escherichia coli have been developed by fusion of antigenic peptides to the flagellar filament (1, 2). There have also been reports on relatively larger proteins being expressed on the cell surface using the outer membrane proteins LamB (3), PhoE (4), OmpA (5, 6) and the peptidoglycan-associatedlipoprotein (PAL) (7). However, there have been few reports on the display of large peptides or proteins including antibodies with E. co/i flagellar filaments (8, 9). A filamentous phage system (10-12) has been developed and used for the expression of peptides as well as of sFv. In this case, the peptides and the proteins are expressed as part of the virus coat protein. One or two of the three coat proteins are displayed on the surface of the phage particles as fusion proteins. In order to develop a novel protein display system in E. coli, we focused on the protein, flagellin (13-15). Flagellin is the constituent protein which assembles to form flagellar filaments on the surface of E. coli. The advantageous aspect of constructing fusion proteins with flagellin is that one flagellar filament is composed of about 20,000 flagellin molecules, if a large portion of these flagellin proteins can be replaced with fusion proteins, detection sensitivity will be significantly higher than that reported for the phage display system. Moreover, flagellin can easily be removed from the cell surface by vortexing or heat treatment. This being the case, it would be easy to purify the produced protein fused with flagellin. Fusion proteins displayed on the cell surface can be concentrated by centrifugation of the cells followed by vortexing or heat treatment, which would release the proteins from the cells. The subunit packing and secondary structure arrangement of the flagellar filament has been analyzed by X* Corresponding author. Abbreviations: SDS-PAGE, gel electrophoresis.

sodium

dodecyl

analysis

Dl , D2 and D3, and although

sulfate-polyacrylamide

500

NOTES

VOL. 86. 1998

A

Fusion protein

Plasmid Dl

D2

03

6 D2

Dl

1

pTF1

1 pTF2-K

c ’

pTF3

~‘,rrr’~

pTF3-K

C

pTF4 pTF4-K

I

pTF2

L

501

2

3

4

5

M

, I

KDa

t -rr,,,

, Lrrrra

-

P,,I,I

7 J 20

-

Peptide linker

-

Flagellin D2 domain

-

Flagellin Dl domain -

Flagellin D3 domain

14

FIG. 1. SDS-PAGE of flagellin-linker fusion proteins. (A) Schematic representation of various recombinant plasmids. (B) SDS-PAGE of fusion proteins. The fusion proteins appeared as one major band in the flagellar fraction. Lanes: 1, flagellar fraction from pTF4K; 2, flagellar fraction from oTF3K: 3. flaaellar fraction from DTFZK: 4. flaaellar fraction from pTF1 harboring native hag; 5, flagellar fraction from host strain I

(YK4516); 6, rbolecuiar’maiers.

fusion protein from plasmid pTF4K showed high levels of fusion protein in the flagellar fraction; at levels higher than those in pTF2K and pTF3K, and equivalent to that in pTF1. Therefore, pTF4K was used as the standard plasmid for further expression of fusion proteins. The sFv and constant region (region C) of the antiporphyrin antibody (20) were used to construct fusion proteins with flagellin. Region C and sFv consisted of 110 and 249 amino acid residues, respectively (Table l), leading to the formation of 65.6- and 80.6-kDa fusion proteins. We observed a large amount of the region C fusion protein in the llagellar fraction (Fig. 2, lane 2). The efficiency was equivalent to that produced by pTF4K which contains the peptide linker (lane 1). Although the amount of fusion protein was less than that of the flagellin-linker protein, we could also detect the flagellin sFv fusion protein in the flagellar fraction (lane 3). We next examined the possibility of displaying the 49.4-kDa bacterial alkaline phosphatase (BAP) (21). The l.Ckbp DNA fragment of the BAP gene from E. coli JM109 was inserted into the central region of the flagellin gene in the plasmids pTF3K and pTF4K to construct pTF3KAK and pTFCKAK, respectively. The fusion proteins from cells harboring pTF3-KAK and pTF4-KAK were analyzed by SDS-PAGE and Western blotting (Fig. 3). We found that most of the fusion proteins (BAP/ Fla) remained in the intracellular fraction. However, we were still able to detect BAP/Fla in the flagellar fraction by staining with Coomassie brilliant blue. Western blot analysis was conducted to confirm that the proteins detected in the flagellar fraction were actually the fusion TABLE 1. Protein Peptide linker Region C GFP Anti-porphyrin antibody sFv Alkaline phosphatase

proteins. The results of expression and display of various flagellin fusion proteins in the present study are shown in Table 1. We were able to display all flagellin fusion proteins with P15, region C, sFv, and BAP. Of the three types of chimeric flagellin (pTF2, pTF3 and pTF4), the pTF4 series produced the highest amount of flagellin fusion protein. Therefore, inserting the foreign proteins into the intact flagellin protein was effective. There was a tendency for fusion proteins with short inserts to be more efficiently transported to the flagellar filament. This was not due to a low efficiency of gene expression of longer fusion genes, as we could detect high levels of intracellular BAP/Fla. We also displayed green fluorescent protein (GFP) (22), which is a novel genetic reporter protein. This protein has a chromophore, consisting of a cyclic tripeptide derived from Ser-Tyr-Gly in the primary protein sequence and yields bright green fluorescence when illuminated by blue light (395 nm) (23). The flagellin-GFP fusion protein (GFP/fla) in the flagellar fraction was detected by SDS-PAGE, and fluorescent micrography showed that the E. coli transformant produced the GFP/fla protein (data not shown). Our results indicate that it was possible to display proteins (up to 49.4 kDa) on the cell surface of E. cofi using flagellin fusion proteins. The system offers some advantages compared to previously reported display systems in E. coli. After cultivation, the fusion proteins can be liberated from the cells by simple vortexing or heat treatment. Intracellular and secreted proteins are not included in the flagellar fraction, enabling easy purification.

Expression of various flagellin fusion proteins

M.W. (kDa) 1.2 11.8 26.9 26.9 49.4

-tt, High level; *, moderate high level; f, moderate level.

Nucleotide (bp) 45 330 714 741 1413

Intracellular expression

Cell surface display

L

LL ..

tc

t t t

t M

502

EZAKI ET AL. M

1

2

J 3

FERMENT. BIOENG.,

heterologous epitopes on Staphylococcus xylosus as a potential delivery system for oral vaccination. Gene, 128, 89-94 (1993).

KDa 212 170

2. Kuwajiia, G., Asaka, J.-I., Fujiwara, T., Fujiwara, T., Nakano, K., and Kondoh, E.: Presentation of an antigenic determinant

76

4

sFv

4

region C

4

from hen egg-white lysozyme on the flagellar filament of Escherichia co/i. BioITechnology, 6, 1080-1083 (1988). 3. Agterberg, M., Adriaanse, H., and Tommassen, J.: Use of outer membrane protein PhoE as a carrier for the transport of a foreign antigenic determinant to the cell surface of Escherichia coli K-12. Gene, 59, 145-150 (1987).

region C

4. O’Callaghan, D., Charbit, A., Martineau, P., Leclerc, C., Werf, S. V. D., Nauciel, C., and Hofnung, M.: Immunogenicity of foreign peptide epitopes expressed in bacterial envelope proteins. Res. Microbial., 141, 963-969 (1990). 5. Francisco, J.A., Earhart, C.F., and Georgiou, G.: Transport

53

SDS-PAGE

Western

and anchoring of P-lactamase to the external surface of Escherichia coli. Proc. Natl. Acad. Sci. USA, 89, 2713-2717 (1992). 6. Francisco, J. A., Campbell, R., Iverson, B. L., and Georgiou, G.: Production and fluorescence-activated cell sorting of Escherichia coli expressing a functional antibody fragment on the external surface. Proc. Natl. Acad. Sci. USA, 90, 10444-10448 (1993). I. Fuchs, P., Weichel, W., Dubel, S., Breitling, F., and Little, M.: Separation of E. coli expressing functional cell-wall bound antibody fragments by FACS. Immunotechnology, 2, 97-102 (1996).

blot

FIG. 2. SDS-PAGE and Western blot analysis of flagellin-antibody fusion proteins. Lanes: M, molecular markers; 1, flagellar fraction from pTF4K; 2, flagellar fraction from pTFCKCK harboring the linker-C region-linker fragment; 3, flagellar fraction from pTF4-KSK harboring the linker--sFv-linker fragment; 4, flagellar fraction from pTFCKCK.

Furthermore, the abundance of flagellin molecules on the cell surface increases the potential to display a significantly larger amount of fusion proteins compared to conventional methods. This has clearly been shown in the case of peptide linkers. For effective expression and display of large flagellin fusion proteins, modification of domain 3 and peptide linker based on the 3D structure of flagellin will be needed in future studies. A practical application of our system would be to determine the epitope regions of an antigen. The high levels of epitopes displayed on the cell surface would lead to easier detection with antibodies. Jast as it was possible to display BAP/Fla on the cell surface, it should be possible to increase the efficiency of display for proteins of larger size through future optimization studies.

8. Westerlund-Wikstrom, B., Tanskanen, J., Virkola, R., Hacker, J., Lindberg, M., Skurnik, M., and Korhonen, T. K.: Func-

tional expression of adhesive peptides as fusions to Escherichia coli flagellin. Protein Eng., 10, 1319-1326 (1997). 9. Lu, Z., Murray, K. S., Cleave, V. V., LaVallie, E. R., Stahl, M. L., and McCoy, J. M.: Expression of thioredoxin random

peptide libraries on the Escherichia coli cell surface as functional fusions to flagellin: a system designed for exploring proteinprotein interactions. Biotechnology, 13, 366-372 (1995). 10. Chiswell, D. J. and McCafferty, J.: Phage antibodies: Will new ‘coliclonal’ antibodies replace monoclonal antibodies? TrendsBiotechnol., 10, SO-84 (1992). 11. Hoogenboom, H. R., Griffiths, A. D., Johnson, K. S., Chiswell, D. J., Hudson, P., and Winter, G.: Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. Nucl. Acids Res., 19, 4133-4137 (1991). 12. McCafferty, J., Fitzgerald, K. J., Earushaw, J., Chiswell, D. J., Link, J., Smith, R., and Kenten, J.: Selection and rapid purification of murine antibody fragments that bind a transition-state analog by phage display. Appl. Biochem. Biotechnol., 47, 157-173 (1994). 13. Kuwajima, G., Asaka, J.-I., Fujiwara, T., Node, K., and Kondo, E.: Nucleotide sequence of the hag gene encoding flagellin of Escherichia co/i. J. Bacterial., 168, 1479-1483 (1986).

This research was supported by the lnstitute for Fundamental Research and Biological Science Laboratories of Kao Corporation, l-3, Bunka 2-chome, Sumida-ku, Tokyo, Japan. REFERENCES

1. Nguyen, N. T., Hansson, Domzig,

M., Stuhl, S., B&hi, T., Robert, A., W., Binz, H., and UhlCn, M.: Cell-surface display of

M

1

2

3

4

3

4

KDa 94 c1 67

SDS-PAGE

Western

blot

FIG. 3. SDS-PAGE and Western blot analysis of BAP/fla proteins. a: BAP/fla protein from pTFCKAK; b: BAP/fla protein from pTF3KAK. Lanes: M, molecular markers; 1, intracellular fraction from pTF3-KAK; 2, intracellular fraction from pTFCKAK; 3, flagellar fraction from pTF3-KAK; 4, flagellar fraction from pTF4-KAK.

VOL. 86, 1998 14. Joys, T. M.: The covalent structure of the phase-l flagellar filament protein of Salmonella typhimurium and its comparison with other flagellins. J. Biol. Chem., 260, 15758-15761 (1985). 15. Ikeda, T., Homma, M., Iino, T., Asakura, S., and Kamiya, R.: Localization and stoichiometry of hook-associated proteins within Salmonella typhimurium flagella. J. Bacterial., 169, 1168-1173 (1987). 16. Namba, K., Yamashita, I., and Vonderviszt, F.: Structure of the core and central channel of bacterial flagella. Nature, 342, 648-654 (1989). 17. Kuwajima, G.: Construction of a minimum-size functional flagellin of Escherichia coli. J. Bacterial., 170, 3305-3309 (1988). 18. Beerli, R. R., Wels, W., and Hynes, N. E.: Intracellular expression of single chain antibodies reverts ErbB-2 transformation. J. Biol. Chem., 269, 23931-23936 (1994). 19. Freund, C., Ross, A., Guth, B., Pluckthun, A., and Holak,

NOTES

503

T. A.: Characterization of the linker peptide of the single-chain Fv fragment of an antibody by NMR spectroscopy. FEBS Lett., 320, 97-100 (1993).

20. Kohda, K., Kakehi, M., Ohtsuji, Y., Tagaki, M., and Imanaka, T.: Studies of high thermostability and peroxidase activity of recombinant antibody L chain-porphyrin Fe(lll) complex. FEBS Lett., 407, 280-284 (1997). 21. Shuttleworth, H., Taylor, J., and Minton, N.: Sequence of the gene for alkaline phosphatase from Escherichia coli JM83. Nucl. Acids Res., 14, 8689-8689 (1986). 22. Chalfie, M., Tu, Y., Euskircheo, G., Ward, W. W., and Prasher, D. C.: Green fluorescent protein as a marker for gene expression. Science, 263, 802-805 (1994). 23. Prasher, D. C., Eckenrode, V. K., Ward, W. W., Prendergast. F. G., and Cormier, M. J.: Primary structure of the Aequorea Victoria green-fluorescent protein. Gene, 111, 229-233 (1992).