재조합 곤충세포에서 생산된 인간 인터루킨-2의

녹색형광단백질과 금속친화배위자와의 융합을 통한 분리정제

차형준, William E. Bentley

포항공과대학교 화학공학과, 메릴랜드 대학교 화학공학과

Purification of Human Interleukin-2 Produced

in Recombinant Insect Cells by Fusion

with Green Fluorescent Protein and Metal Affinity Ligand

Hyung Joon Cha and William E. Bentley

Dept. of Chemical Engineering, Pohang University of Science and Technology and Dept. of Chemical Engineering, University of Maryland at College Park

Abstract

Fusion protein of green fluorescent protein (GFP) and human interleukin-2 (hIL-2) was produced in insect Trichoplusia ni larvae infected with recombinant baculovirus derived from the Autographa californica nuclear polyhedrosis virus (AcNPV). This fusion protein was composed of a metal ion binding site (His)6 for rapid one-step purification using immobilized metal affinity chromatography (IMAC), UV-optimized GFP (GFPuv), enterokinase cleavage site for recovering hIL-2 from purified fusion protein, and hIL-2 protein. The additional histidine residues on fusion protein enabled the efficient purification of fusion protein based on IMAC. Addition to advantages of GFP as a fusion marker, GFP was able to be used as a selectable purification marker; we easily determined the correct purified fusion protein sample fraction by simply detecting GFP fluorescence.

Introduction

A fusion protein consisting of an histidine affinity ligand, the green fluorescent protein, an enterokinase cleavage site linker, and hIL-2 was expressed in insect (Trichoplusia ni) larvae. This fusion construct enabled facile detection and one-step purification of hIL-2 fusion using immobilized metal affinity chromatography. By incorporating the gene for the GFP (Chalfie et al., 1994) into the baculovirus expression vector, a marker can be created for visualizing gene expression (Cha et al., 1997; Cha et al., 1999a), product separation, and purification in real time. An N-terminal affinity ligand (His)6 makes GFPuv amenable to purification with IMAC. This histidine ligand has previously been demonstrated for purification of viral proteins expressed in the insect cell / baculovirus expression system (Wang et al., 1994). Since histidine is not a common residue in protein sequences, and accounts for only 2.1% of amino acids in globular proteins (Klapper, 1977), there is a high probability that a desired protein engineered to contain additional histidines on its N- or C-terminus, will be preferentially separated from other proteins. This technique has gained popularity in recent years (Hale and Beidler, 1994; Chaga et al., 1994).

The final component of the fusion protein is a 25 amino acid linker sequence between the C-terminus of the GFPuv and the N-terminus of hIL-2. Within this sequence is a 5-residue enterokinase (EK) cleavage site so that the GFPuv, the (His)6 affinity ligand, and the EK cleavage linker can be removed enzymatically from the hIL-2. Hence, the goal of the present effort was to create a multicomponent fusion protein containing a marker, an affinity ligand, and a cleavage site, to develop an integrated processing approach that is simple, amenable for inclusion in rapid protein expression screening programs, and that can be characterized at each step by simple visual inspection.

Materials and Methods

The cabbage looper, T. ni, insect larvae were hatched from eggs in Styrofoam cups containing solid food at 30oC; fourth instar larvae were used for infection experiments. The recombinant baculovirus, vP10-GFPuv/hIL2, encoding GFPuv and hIL-2 fusion protein was constructed as described (Cha et al., 1999b). The recombinant baculovirus, vP10-GFPuv/hIL2 of 1 x 108 pfu/cup was spread on the media which was obtained pre-made in Styrofoam cups. The cups were covered and allowed to stand for 1 h. so that the virus was completely absorbed by the media. The to-be-infected larvae were placed into cups (15 larvae per cup), which were then inverted. The larvae were allowed to feed on the infected food at 30oC.

The total protein assay was performed using an 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 histidine tagged GFPuv and hIL-2 fusion protein was purified by IMAC using a Ni2+ immobilized resin. The column was equilibrated with start phosphate buffer (20 mM phosphate, 0.5 M NaCl, 10 mM Imidazole, pH 7.4) and the homogenized / clarified sample was applied. Several fractions (0.5 mL) were collected as a flow through. The column was washed with start phosphate buffer and sample fractions were collected as a wash sample. Fusion proteins were eluted by elution phosphate buffer of different imidazole concentration (50 mM, 100 mM, 200 mM, 300 mM, 400 mM, and 500 mM). Fractions were monitored at A280 using the corresponding elution buffer as blank and also detected by fluorescence intensity using a fluorescence spectrometer. Most bound proteins were eluted at imidazole concentration between 100-200 mM.

Results and Discussion

Purification of GFPuv and hIL-2 fusion protein was performed using an IMAC column as described in Methods (Figure 1). Fractions collected during the IMAC elution were measured for absorbance at 280 nm (A280) and GFPuv fluorescence intensity. Weakly bound proteins were quickly washed away (fractions 4-23). Most tightly bound proteins were eluted at an imidazole concentration between 100-200 mM (fractions 39-47). In the case of the A280 profile, several peaks were shown (Figure 1A), however, the profile of fluorescence intensity showed a single sharp peak (Figure 1B). By examining fluorescence or more importantly, fluorescence / A280, one can immediately see the relative quantity of GFPuv-containing product in the fraction. That is, determining the product-containing fractions consisted of either checking the GFPuv fluorescence intensity using a fluorescence spectrometer (in our case), or using a fluorescence detector on the chromatography unit, or by simply visualizing the fractions in glass or translucent microfuge tubes while placed on a UV table.

With this GFP-

fusion technique,

difficult and time-

consuming assays

(e.g., Western blot, ELISA, or

enzymatic activity

assays) for selecting

the correct fraction can be omitted.

The eluted

purified samples

were then assayed

by SDS-PAGE and

Western blot. The

purified sample in

the silver stained

SDS-PAGE shows

three major bands

including the fusion

protein (~52 kDa),

partially degraded

fusion protein

containing (His)6,

and GFPuv

containing (His)6.

The GFPuv band is present at higher density than that of the fusion. This was due to non-specific cleavage at the enterokinase cleavage site during the larvae culture (Cha et al., 1999b). Interestingly, in suspended insect cell culture (Sf-9 cells) we again found this non-specific cleavage but degree of cleavage was much lower than in the insect T. ni larvae (Cha et al., 1999a). Recently, we expressed the GFPuv/hIL-2 fusion protein in yeast Pichia pastoris and saw no evidence for non-specific cleavage at the enterokinase cleavage site (not shown). Thus, we suspected that insect cells express an enterokinase or enterokinase-like protease. A more detailed follow-up investigation is underway using an enterokinase specific substrate. When scanning SDS-PAGE gel, the (His)6-tagged GFP bands (including fusion and partially degraded fusion) comprised approximately 95% of the total protein. This represents the purity of the purified sample by IMAC, demonstrating one-step purification of the (His)6-tagged material (analogous to our previous report in Sf-9 cell culture (Wang et al., 1994)).

The original soluble cell lysate sample in the Western blot showed several bands including the fusion protein, some degraded fusion protein, hIL-2 (~18 kDa), and some degraded hIL-2. A polyclonal anti-hIL-2 antibody was used for detection. Again, the appearance of hIL-2 band at the correct molecular weight indicated that a fraction of the fusion protein was non-specifically cleaved at the enterokinase cleavage site. In the case of the eluted purified sample, only the fusion protein was found. After this one-step IMAC purification, 20.8% of the GFPuv fluorescence intensity was recovered overall, and according to our Western blot data about 28% of the hIL-2 was lost due to the non-specific cleavage and non-binding of the hIL-2 so that overall, the yield of hIL-2 was 19.8%. The purification ratio, based on captured GFP fluorescence, was 35 demonstrating very effective purification of histidine tagged GFP fusion in one step.

References

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Cha, H.J., Dalal, N.G., Vakharia, V.N., and Bentley, W.E., J. Biotechnol. 69, 9-17 (1999a).

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Chaga, G., Widersten, M., Andersson, L., Parath, J., Danielson, U., and Mannervik, B., Protein Eng. 7, 1115-1119 (1994).

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