High-level production of human leptin by fed-batch cultivation of recombinant Escherichia coli and its purification

Abstract

Human leptin is a 16-kDa (146-amino-acid) protein that is secreted from adipocytes and influences body weight homeostasis. In order to obtain high-level production of leptin, the human obese gene coding for leptin was expressed in Escherichia coli BL21(DE3) under the strong inducible T7 promoter. The recombinant leptin was produced as inclusion bodies in E. coli, and the recombinant leptin content was as high as 54% of the total protein content. For production of recombinant human leptin in large amounts, pH-stat fed-batch cultures were grown. Expression of leptin was induced at three different cell optical densities at 600 nm (OD600), 30, 90, and 140. When cells were induced at an OD600 of 90, the amount of leptin produced was 9.7 g/liter (37% of the total protein). After simple purification steps consisting of inclusion body isolation, denaturation and refolding, and anion-exchange chromatography, 144.9 mg of leptin that was more than 90% pure was obtained from a 50-ml culture, and the recovery yield was 41.1%.

FIG. 1

(A) Schematic diagram of plasmid pEDOb5. The PCR product (length, 438 bp) which encodes mature human leptin was digested with NdeI and BamHI and then cloned into pET21c at the same restriction sites. (B) Diagram illustrating the main features of pEDOb5. Expression of the human obese gene is controlled by the T7 promoter (P_T7). A tandem repeat of the stop codon follows the human obese gene. RBS, ribosome binding site.

FIG. 2

SDS-PAGE analysis of leptin produced by a flask culture. Each fraction corresponds to 10 μl of protein homogenate. Lane 1, molecular mass standards; lane 2, total proteins; lane 3, inclusion body fraction; lane 4, soluble protein fraction.

FIG. 3

Time profiles for cell density (OD₆₀₀) (■), cell dry weight (○), and leptin content (▴) during fed-batch cultivation with induction at the low cell density (A), the intermediate cell density (B), and the high cell density (C). The dashed lines indicate the time of induction.

FIG. 4

SDS-PAGE analysis of samples from each purification step. Lane 1, molecular mass standards; lane 2, inclusion body fraction after washing with double-distilled H₂O; lane 3, supernatant after denaturation and centrifugation; lane 4, refolded leptin after dialysis; lane 5, sample after anion-exchange chromatography.

FIG. 5

Mass spectrum of purified recombinant human leptin. The matrix-assisted laser desorption-ionization mass spectrum is dominated by a single component (leptin, the second peak) with a measured molecular mass of 16,149.1. The first peak (8,074.81) represents the doubly protonated form of the protein arising from the matrix-assisted laser desorption-ionization mass spectrometric process. The third peak (32,303.6) represents a leptin dimer.

FIG. 6

Electrophoretic analysis of the redox state of leptin after treatment with a reducing agent (5 mM DTT). Lane 1, molecular mass standards; lane 2, leptin not treated with DTT; lane 3, DTT-treated leptin.