재조합 대장균 배양에서 fusion partner로서의

green Fluorescent protein을 이용한 fusion 단백질의 발현

차형준, Chi-Fang Wu, William E. Bentley

메릴랜드대학교 화학공학과

Expression of Fusion Protein using Green Fluorescent Protein

as a Fusion Partner in Recombinant Escherichia coli

Hyung Joon Cha, Chi-Fang Wu, William E. Bentley

Department of Chemical Engineering, University of Maryland

Introduction

In our previous work, we demonstrated the use of green fluorescent protein (GFP) as a quantitative marker of protein level in the insect expression system (Cha et al., 1997; Cha et al., 1999). In the present work, we demonstrate the use of GFP as a non-invasive quantitative fusion marker of foreign protein production in recombinant E. coli. Unique attributes of GFP as a protein marker are: (1) it requires no co-factors or staining for fluorescence and (2) the fluorescence is readily visible from outside the cells (Chalfie et al., 1994). We have chosen human interleukin-2 (hIL-2), CAT, and organophosphorous hydrolase (OPH) as three model products. Because of its role in tumor attacking lymphocyte proliferation, IL-2 has been evaluated as a therapeutic agent in the treatment of cancer (Rosenburg et al., 1984). Since hIL-2 is very insoluble, it was previously expressed as insoluble aggregates of inactive protein in recombinant E. coli, even though it was biologically active after purification. Chloramphenicol acetyl-transferase, CAT, is a bacterial protein that confers antibiotic resistance to chloramphenicol. It is widely used as a marker because its enzymatic activity can be reliably assayed in several ways (Rodriquez and Tait, 1983). Organophosphorous hydrolase, OPH, is a homodimeric enzyme that can degrade a broad spectrum of organophosphorus pesticides such as paraoxon, parathion, coumaphos, and diazinon. It can also hydrolyze many chemical warfare agents such as soman, sarin, and VX (O-ethyl S-(2-diisopropylaminoethyl) methylphosphonothiolate) (Grimsley et al., 1997).

Materials and Methods

E. coli TOP10 was used for constructing recombinant plasmid containing the GFPuv and hIL-2 genes. E. coli BL21 and JM105 were used for expressing the fusion proteins. Recombinant E. coli strains were grown to mid-exponential phase (at ~ OD600 = 0.6) at 37oC in 200 mL of LB medium containing 50 mg/mL ampicillin. These cultures were inoculated (5% (v/v)) from 37oC overnight cultures in the same medium. At OD600 = 0.6, E. coli strains were induced by the addition of 1 mM IPTG to express the fusion protein.

Cell growth was monitored by optical density (at 600 nm, OD600) on a UV/VIS spectrophotometer. The total protein assay was performed using a protein assay kit with bovine serum albumin as a standard. The GFPuv assay was performed by measuring fluorescence intensity using a fluorescence spectrometer at an excitation wavelength of 395 nm and emission at 509 nm. The quantities of hIL-2 and CAT were determined using pure recombinant hIL-2 and pure CAT from E. coli  as a calibration standard on Western blots. In the case of OPH, the standard was made via purification using immobilized metal affinity chromatography (IMAC) in our laboratory. The CAT activity assay was performed using a UV/VIS spectrophotometer at 37oC as described by Rodriguez and Tait (1983); samples with high activity were diluted with deionized water to obtain readings in the linear range. The units of CAT activity were defined as nmoles/min. Specific CAT activity was reported as units/(mg-CAT). The OPH activity assay was performed using a UV/VIS spectrophotometer. The absorbance change was monitored at a wavelength of 400 nm. The units of OPH activity were defined as mmoles of paraoxon hydrolyzed per min.

Results and Discussion

Figure 1 shows the photo of fusion protein expressing recombinant E. coli. Cells were photographed using fluorescent microscopy. E. coli BL21 [pTH-GFPuv/CAT] emitted less green fluorescence (Fig. 1A) than that of BL21 [pGFPuv] which expressed only the gfpuv gene instead of a fusion (Fig. 1B). However, the fusion- containing cells fluoresced implying the GFPuv/CAT fusion protein was expressed. We could also observe the green fluorescence from cells grown on agar plates using a UV transilluminator for excitation (not shown). These results demonstrated that the GFP fusion technique enabled us to clearly ascertain the expression of desired proteins in recombinant E. coli.

In Figure 2, we demonstrated that the GFP-product fusions were expressed at the appropriate molecular weights for all host cells and for all products (CAT, hIL-2, OPH). Interestingly, the GFPuv whole cell fluorescence was barely detected in pTH-GFPuv/hIL2 containing strains (not shown), while from the Western blot analysis, we obtained the proper GFPuv/hIL-2 fusion band (~52 kDa, lanes 1S - 2IS). Since the Western analysis confirmed expression, we suspected that most of the GFPuv/hIL-2 fusion protein was expressed as inactive inclusion bodies. This was confirmed by comparing the insouble to the soluble fractions, and it likely due to the relative insolubility of hIL-2; all published on hIL-2 expression from E. coli culture have been similar in this respect (Devos et al., 1983).

Linear regression analyses of GFPuv fluorescence intensity with the concentration of the product proteins (CAT, OPH, hIL-2) and enzymatic activity (CAT, OPH) was performed. Because of the time lag for GFPuv chromophore cyclization, which has been shown to be constant, irrespective of fermentation condition, (~95 min; Crameri et al., 1996), the GFPuv fluorescence intensity was shifted 95 min. Linear correlations were obtained in both amount and activity for both GFPuv/CAT and GFPuv/OPH samples. Interestingly, the correlation between fluorescence and CAT was obtained for samples of two different E. coli strains BL21 and JM105, demonstrating that the correlation was independent of the host strain. One observation, however, was that the slope of the correlation was typically fusion partner dependent. That is, one would need to run a calibration experiment to obtain the fluorescence signal that correlates with product quantity, as opposed to wrote monitoring GFP fluorescence. Again, hIL-2. a linear correlations obtained but because of the low level, it was less significant.

References

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