재조합 대장균에서의 Human Leptin 생산에 따른 프로테옴 분석

한미정, 최종현, 정기준, 유종신*, 이상엽

대전광역시 유성구 구성동 373-1 한국과학기술원 화학공학과 및 생물공정연구센터, 대전광역시 유성구 어은동 52 기초과학지원연구소*

Proteome Analysis of Overproduction of Human Leptin

in Escherichia coli by Fed-Batch Fermentations

Mee-Jung Han, Jong Hyun Choi, Ki Jun Jeong,

Jong-Shin Yoo*, Sang Yup Lee

Department of Chemical Engineering and BioProcess Engineering Research Center, KAIST 373-1 Kusong-dong, Yusong-gu, Taejon 305-701, Korea Basic Science Institute, 52 Yeoeun-dong Yusong-gu, Taejon 305-333, Korea*

Introduction

Escherichia coli is one of the most widely used hosts for the production of heterologous proteins and its genetics are far better characterized than those of any other microorganism. The formation of intracellular protein aggregates, referred to as inclusion bodies (IBs), is a phenomenon often accompanying the expression of recombinant proteins in E. coli. Inclusion body formation is not limited to damaged, mutant or foreign proteins. Altered conditions of expression of the wild type E. coli proteins within the normal host can also result in inclusion body formation. Importantly, recombinant protein overexpression results in a rapid □stress□ response. A key feature of this response is increased protease activity.  Additional features include increased plasmid instability and cell lysis and decreased growth rate. This response is generally referred to as the □metabolic burden□ associated with the production of a nonessential protein. The phenotypic characteristics of this response are similar to the responses E. coli have developed to heat shock and amino acid limitation. Interestingly, the stress response of E. coli includes an increase in genetic variation (transposition events) which is presumed to provide the host with a tool for adaptation to environmental changes, a feature not desired when maintaining reproducibility among repeated fermentations. Several studies indicate an increase in the levels of heat shock proteins like DnaK, GroEL, and GrpE after overexpression of heterologous proteins. The heat shock-like response during heterologous protein expression may also enhance intracellular proteolytic activities. Furthermore, host stress proteins, like DnaK, GroEL, IbpA, IbpB, and OmpT, have been reported to be associated with the protein aggregates (3).

In this paper, the cellular response to overproduction of human leptin by recombinant E. coli in high-cell-density cultures is investigated by 2-gels and MALDI-TOF mass spectrometry. The recombinant human leptin was produced as inclusion bodies in E. coli, and the recombinant human leptin content was as high as 30% of the total protein content. Furthermore, the insoluble human leptin protein pattern was determined by 2DE.

Materials and methods

Cell culture conditions

E. coli BL21(DE3) was used as a host strain for expression of the obese gene. The mature obese gene, pEDOb5 was expressed from the strong T7 promoter by induction with isopropyl-□-D-thiogalactopyranoside (IPTG). Cells were cultivated in 250-ml flasks containing 50 ml of Luria-Bertani (LB) medium supplemented with 50 □g of ampicillin per ml in a shaking incubator at 37□C and 200 rpm as the same procedures (2). Fed-batch cultures were grown in a 6.6-liter jar fermentor (Bioflo 3000; New Brunswick Scientific Co., Edison, N.J.) containing 1.8 liters of R/2 medium. Expression of the obese gene was induced by adding IPTG to a final concentration of 1 mM.

One-dimensional gel electrophoresis

Cells were harvested by centrifugation at 6,000 □ g for 10 min at 4□C. The cells were disrupted by sonication (Branson Ultrasonics Co., Danbury, Conn.) for 1 min at 40% output. After centrifugation at 10,000 □ g for 10 min at 4□C, the supernatant fluid (soluble protein fraction) was saved, and the pellet (inclusion body fraction) was resuspended in 10 mM Tris-HCl buffer (pH 8.0).

Two-dimensional gel electrophoresis (2-DE)

The 2-DE was carried out using Protean II xi 2-D Cell (Bio-Rad Laboratories, Hercules, CA) following the procedures described previously with slight modifications as follows (1). Culture broth was centrifuged for 5 min at 3,500 × g and 4℃. The pellet was washed 4 times with low salting washing sample buffer (3 mM KCl, 1.5 mM KH2PO4, 68 mM NaCl, 9 mM NaH2PO4) and was then resuspended in a buffer containing 10 mM Tris-HCl pH 8.0, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol (DTT), 0.5 mM Pefabloc SC, 0.1% (w/v) sodium dodecyl sulfate (SDS), and stored at -20 ℃. 3 □l of the latter was mixed with 60 □l of a solution containing 8 M Urea, 4% (w/v) 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), 40 mM Tris, 65 mM DTT and was centrifuged for 10 min at 10,000 × g and 15℃. After the protein quantification by Bradford assay using bovine serum albumin, protein samples (300 □) were resuspended in IEF denaturation buffer [9 M Urea, 0.5% (w/v) CHAPS 10 mM DTT, 0.2% (w/v) Bio-lyte pH 3-10, 0.001% (w/v) Bromophenol Blue], and the whole final diluted sample was loaded onto the immobilised pH gradient (IPG) strips pH4-7, 17 cm. Isoelectric-focusing (IEF) was performed using PROTEAN IEF CELL (Bio-Rad Laboratories, Hercules, CA) by a step-wise protocol for 60,000 vh for IPG strips 17 cm. The strips were placed in equilibration buffer I [6 M urea, 2% (w/v) SDS, 0.375 M Tris-HCl, pH 8.8, 20% (v/v) glycerol, and 130 mM DTT] for 15 min and next in buffer II (6 M urea, 2% (w/v) SDS, 0.375 M Tris-HCl, pH 8.8, 20% (v/v) glycerol, 135 mM iodoacetamide and 3.5 M bromophenol blue) for 15 min. Subsequently, the loaded strips were placed onto 12% SDS-PAGE gels prepared by a standard protocol. Protein was visualized by PowerStain gel staining system (Elpis-Biotech, Korea). This staining system based on Coomassie brilliant blue R-250 allows detection of most proteins down to the nanogram range, which is nearly similar to sensitivity of silver staining. These gels were scanned using GS710 Calibrated Imaging Densitometer (Bio-Rad Laboratories, Hercules, CA). Melanie II software (Bio-Rad Laboratories, Hercules, CA) was used to automate the process of finding protein spots within the image and to quantify the density of the spots in volume basis (i.e. values were calculated from the integration of spot optical intensity over the spot area). Each 2-DE gel was matched with E. coli SWISS 2DPAGE and identified proteins by MALDI-TOF mass spectrometer. To check the reproducibility and to estimate standard deviation, protein samples taken from duplicate cultures were analyzed in duplicate 2-DE gels.

Results and discussion

Fed-batch cultivation

Fed-batch culture of E. coli BL21(DE3) harboring pEDOb5 was grown as described above. E. coli BL21(DE3) without the plasmid and recombinant E. coli BL21(DE3)(pED) were also carried out as controls. The time profile of E. coli BL21(DE3)(pEDOb5) was shown Figure 1.

Expressed protein profiles

We prepared for 4 samples at the nearly same concentration from E. coli BL21(DE3)(pEDOb5) and controls, respectively. Proteome expression profile of insoluble proteins is shown in Figure 2. From two independent experiments, overall proteome profiles are reproducible, and spots are quite distinctive enough to be matched and compared quantitatively. We matched with SWISS 2DPAGE E. coli master gel, and database of our laboratory and resulted in identifying with good certainty on 2D-gels. Density-derived variation of the protein expression level was verified by the software-aided quantification. From a number of identifiable protein spots, we were able to understand global physiological changes for overproduction of the recombinant model protein and this valuable information would be utilized towards the strain improvement and the construction of various databases regarding metabolism.

Figure 2. Proteome profile of insoluble protein of E. coli BL21(DE3)(pEDOb5).

Figure . The time profile of E. coli            BL21(DE3)(pEDOb5).

Acknowledgement

This work was supported by the Ministry of Commerce, Industry and Energy through Korea Institute of the Industrial Technology Evaluation and Planning (ITEP).

Referances

1. Hochstrasser, D. F., Harrington, M. G., Hochstrasser, A. C., Miller, M. J. and Merril, C. R., "Methods for increasing the resolution of two-dimensional protein electrophoresis" Anal. Biochem., 173, 424(1988).

2. Jeong, K. J. and Lee, S. Y., "High-level production of human leptin by fed-batch cultivation of recombinant Escherichia coli and its purification" Appl. Environ. Microbiol., 65, 3027(1999).

3. Jurgen, B., Lin, H. Y., Riemschneider, S., Scharf, C., Neubauer, P., Schmid, R., Hecker, M. and Schweder, T., "Monitoring of genes that respond to overproduction of an insoluble recombinant protein in Escherichia coli glucose-limited fed-batch fermentations" Biotechnol. Bioeng., 70, 217(2000).