재조합 대장균에서 DsbA 동시발현을 이용한

인체 유래 leptin 단백질의  분비생산

정기준, 이상엽

대전광역시 유성구 구성동 373-1 한국과학기술원 화학공학과 및 생물공정연구센터

Production of soluble human leptin in periplasmic space of Escherichia coli using DsbA coexpression

Ki Jun Jeong and Sang Yup Lee

Department of Chemical Engineering and BioProcess Engineering Research Center, KAIST 373-1 Kusong-dong, Yusong-gu, Taejon 305-701

Introduction

Obesity is a major risk factor for the development of chronic diseases such as hypertension, type II diabetes and hyperlipidermia. Human leptin, the product of obese gene, is a 16 kDa (146 amino acid) protein secreted from adipocytes into bloodstream and has recently been identified as one of the factors controlling satiety. Administration of recombinant leptin to ob/ob mice, which are deficient in the production of leptin, cause a reduction in food intake and weight loss.  The functions of leptin in regulating appetite and metabolism as well as the possibility of using it as a therapeutic agent are currently under intense investigation. Further detailed investigation of the function of leptin or its use as a potential therapeutic intervention in obesity depends on the availability of a highly pure, safe, biologically active product. It has been reported that the leptin refolded from inclusion bodies was remarkably less potent than that secreted from eukaryotic cells, and furthermore, the biological potency of leptin varied considerably depending on the refolding methods employed. However, the soluble leptin secreted into the periplasm of E. coli showed similar biological potency to native leptin. It is suggested that the difference in the potency of secreted versus refolded leptin may rest in difference in the conformation of refolded protein. To date, however, secretory production of leptin in E. coli was rather inefficient.

We demonstrate efficient secretion of human leptin into the periplasm of E. coli using endoxylanase signal peptide, a new signal peptide cloned from Bacillus sp. We also demonstrate the successful use of oxidoreductase DsbA for increasing the solubility of human leptin under normal cultivation condition

Materials and methods

1. Bacterial strains and plasmids

E. coli strains	Relevant characteristics
XL1-Blue	SupE44 hsdR17 recA1 endA1 gyrA96 thi relA1 lac F(proAB+ lacIq lacZM15 Tn10(tetr))
BL21(DE3)	F- ompT hsdSB (rB- mB-) gal dcm (DE3)
plasmid
pEDSOb4	6.0 kb, Apr, obese gene fused to endoxylanase signal sequence, T7 promoter
pTrcSOb4	4.8 kb, Apr, obese gene fused to endoxylanase signal sequence, Trc promoter
pTrcKOb4	5.1 kb, Kmr, obese gene fused to endoxylanase signal sequence, Trc promoter
pTrcKObD	6.0 kb, Kmr, obese gene fused to endoxylanase signal sequence, Trc promoter, DsbA co-expression

2. Culture Conditions

For flask cultures, LB medium (tryptone 10 g/L, yeast extract 5 g/L, NaCl 5 g/L) was used. Cells were cultivated in 250 mL flask containing 50 mL LB medium supplemented with 50 g/mL ampicillin (Ap) or 25 g/mL kanamycin (Km) in a shaking incubator at 37℃ or 30℃ and 200 rpm. At the OD600 of 0.7, IPTG was added to a final concentration of 0.05 or 1.0 mM, and cells were further cultivated for 6 h.

3. Cell fractionations

Cells from 1 mL culture broth were harvested by centrifugation at 3,500g for 10 min at 4℃. For the fractionation of soluble and insoluble proteins, cells were disrupted by sonication. After centrifugation at 10,000g for 10 min at 4oC, the supernatant (soluble protein fraction) was saved and the pellet (inclusion body fraction) was resuspended in 10 mM Tris-HCl (pH 8.0) buffer. The periplasmic proteins were prepared by modified osmotic shock procedure.

4. Analitical methods

Cell growth was monitored by measuring the absorbance at 600 nm (OD600). Protein samples were analyzed by electrophoresis on a 12% (w/v) sodium dodecyl sulfate polyacrylamide gel as described by Laemmli. The protein bands on the SDS-PAGE gels were quantified by densitometry.

Results and discussion

1. Secretion of human leptin using T7 promoter

E. coli BL21(DE3) harboring pEDSOb4 were grown in LB medium and were induced with 1 mM IPTG. Most of human leptin was produced as premature form (19 kDa), which indicates that leptin was not secreted into periplasm.

2. Secretion of human leptin using Trc promoter

In E. coli BL21(DE3) harboring pTrcSOb4, human leptin was processed completely (16kDa) with high content (40% of total proteins). However, most of leptin formed inclusion bodies in the periplasm and the secreted soluble leptin could not be found in the periplasmic fraction (그림 1).

3. Localization and N-terminal amino-acid sequence of inclusion body

To examine if leptin was truly secreted into the periplasm, inclusion bodies in the cells were analyzed by transmission electron microscopy. This analysis demonstrated that the inclusion bodies formed in cells harboring pTrcSOb4 were located in the periplasm of E. coli (그림 2). To confirm the correct processing of the signal peptide, N-terminal amino acid sequence of inclusion bodies was analyzed. The determined sequence was Val-Pro-Ile-Gln-Lys- Val-Gln-Asp-Asp, which is consistent with the N-terminal amino acid sequence of mature human leptin. These results indicate that the endoxylanase signal peptide was precisely processed.

4. Production of soluble human leptin by DsbA coexpression

To produce the soluble leptin under the optimal leptin production condition (37oC and 1 mM IPTG induction), the periplasmic oxidoreductase DsbA was co-expressed. In E. coli BL21(DE3) harboring pTrcKObD, the fraction of inclusion body was much lower (그림 3). At 6 h after induction, the content of leptin in the total proteins reached 39% of total proteins, which was similar to that obtained using pTrcKOb4 (Table 2). However, the content of soluble leptin was 26% of total proteins (69% of total leptin), which is much higher than that obtained without DsbA coexpression. The SDS-PAGE of the samples after fractionation of periplasmic proteins revealed a mature leptin as a major band.

Acknowledgement

We thank J. M. Friedman for providing us with the phage construct containing the human obese gene. This work was supported by the Korean Ministry of Commerce, Industry and Energy (MOCIE) through the Institute of Industrial Technology Evaluation and Planning (ITEP).

Referances

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Table 2. Comparision of soluble leptin in E. coli BL21(DE3) harboring pTrcSOb4, pTrcKOb4, pTrcKObD (culture tem. :37℃, Induction with 1 mM IPTG)

plasmid	Leptin in total proteins (%)	Soluble leptin in total leptin (%)	Soluble leptin in total proteins (%)
pTrcSOb4	41±0.8	8.5±0.9	3.5±0.4
pTrcKOb4	40±0.9	9.4±1.3	3.8±0.4
pTrcKObD	38±1.3	69±1.0	26±0.4

그림 1. SDS-PAGE analysis of human leptin produced in BL21(DE3) (pTrcSOb4) by inducing with 1 mM IPTG. Lane 1, molecular mass standard; lane 2, total proteins; lane 3, insoluble proteins; lane 4, soluble proteins.

그림 2. Transmission electron microscopy of E. coli BL21(DE3) expressing human leptin

그림 3. Time profiles of soluble and insoluble human leptin from E. coli BL21(DE3) harboring (A) pTrcKOb4 and (B) pTrcKObD. Lane 1, E. coli BL21(DE3) (no plasmid); lanes 2, 4, 6, 8, and 10, soluble leptins; lane 3, 5, 7, 9, and 11, insoluble leptins