Protein Expression and PuriWcation 46 (2006) 285–293 www.elsevier.com/locate/yprep 1046-5928/$ - see front matter  2005 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2005.08.019 Expression and puriWcation of human placenta lactogen in Escherichia coli Pei Ching Lan, Chi Feng Tseng, Meng Chia Lin, C. Allen Chang ¤ Department of Biological Science and Technology, National Chiao Tung University, 75 Po-Ai Street, Hsinchu, Taiwan, ROC Received 28 June 2005, and in revised form 23 August 2005 Available online 21 September 2005 Abstract There are many growth factors secreted by placenta including growth hormone, placenta lactogen (PL), prolactin, follicle stimulating hormone, luteinizing hormone, thyroid stimulating hormone, and chorionic gonadotropin. For a systematic study of how these growth factors work together to result in the various biological functions and future clinical applications, it is needed to produce enough quanti- ties of each protein. In this paper, we report the cloning of human PL (hPL) and expression by Escherichia coli (E. coli). Four kinds of expression vectors containing the hPL gene were transformed into several kinds of suitable host strains and grown at 37 and/or 30 °C. Determination of the yield of recombinant hPL by SDS–PAGE reveals that among the various conditions, pQE30-PL in E. coli strain M15[pREP4] expressed the largest amount of recombinant hPL at 37 °C. However, the expressed recombinant hPL was accumulated in inclusion body forms. The inclusion bodies were solubilized in 8 M urea and puriWed by a His 6 tagged aYnity column under denaturing condition and the Wnal yield of hPL was determined to be 48 mg/L. Intra-chain disulWde bonds could be formed either by oxidation in the refolding buVer or by air oxidation in the presence of urea. The biological activity was examined by the fact that hPL could stimulate ery- throid maturation by the formation of hemoglobin in K-562 cells in the presence of erythropoietin. Initial optimization studies resulted in the production of 282.4 mg/L of hPL.  2005 Elsevier Inc. All rights reserved. Keywords: Human placental lactogen; Molecular cloning; Protein expression Placenta is an endocrine organ and its extraction mix has been used as precious Chinese medicine [1]. It is known that placenta secretes various immune proteins, thromboplastin, hormones, and many trace substances [2]. These substances collectively adjust body endocrine secretion functions and promote metabolic activities. Many growth factors are secreted by placenta including growth hormone (GH) 1 , pla- centa lactogen (PL), prolactin (Prl), follicle stimulating hor- mone (FSH), luteinizing hormone (LH), thyroid stimulating hormone (TSH), and chorionic gonadotropin (CG). These placental hormones are important. For example, LH, FSH, and CG are also excreted by pituitary and are termed gonadotropins, which help the gonad cells to pro- vide the hormonal milieu and essential factors for germ cell development and intra-testicular cell–cell interactions. These functions are maintained and modulated by other gonadotropins such as GH and Prl [3,4]. On the other hand, the detailed functions of hGH, hPL, and hPrl during preg- nancy are still a matter of debate, but they may have signiW- cant inXuence on the carbohydrate and lipid metabolism of * Corresponding author. Fax: +886 3 572 9288. E-mail address: changca@cc.nctu.edu.tw (C.A. Chang). 1 Abbreviations used: GH, growth hormone; PL, placenta lactogen; Prl, prolactin; FSH, follicle stimulating hormone; LH, luteinizing hormone; TSH, thyroid stimulating hormone; CG, chorionic gonadotropin; hPL, human PL; hGHR, human growth hormone receptor; hGHbp, human growth hormone binding protein; PRLR, prolactin receptor; PCR, polymerase chain reaction; DAF, 2,7-diaminoXourene; SDS–PAGE, Sodium dodecyl sulfate–polyacryl- amide gel electrophoresis; NTA, nitrilotriacetic acid.