以大腸桿菌表現與再折疊重組人類胎盤促乳激素之最適化研究

Abstract

胎盤促乳激素(placental lactogen)是由胎盤中融合層滋養母細胞(syncytiotrophoblast)所分泌，分泌量隨懷孕期間而漸增，在三三月期(third trimester)時，每天分泌量更可達到1-3克。然而關於胎盤促乳激素在懷孕期間所扮演之生理作用和作用機制，以及其合成及分泌是如何調節，到目前為止暸解相當有限。儘管胎盤促乳激素之全部功能仍不明暸，但其已被證實能藉由調控母體代謝的情況，進而影響胎兒的生長，促進母體乳腺上皮細胞的發育。 本實驗的目的在於利用大腸桿菌大量製造recombinant hPL，以提供後續生物或臨床研究所需。實驗中探討影響表現的因子，如啟動子、宿主菌株、溫度、誘導劑濃度以及post-induction time對重組蛋白質表現的影響，結果發現pQE30-PL M15[pREP4]在37℃下，能表現最大量的recombinant hPL，然而大量表現的結果卻造成重組蛋白質以inclusion body的形式存在，經8M urea溶解後，利用His6 tagged affinity column將recombinant hPL純化出來後，經再折疊並對其進行生物活性測試，結果成功證明所獲得的recombinant hPL具有生物活性。

Placental lactogen (PL) is secreted by the placental syncytiotrophoblast in increasing amounts during pregnancy, reaching levels of 1-3 g per day in the last trimester. It has been shown that human placental lactogen (hPL) could influence maternal intermediary metabolism and stimulate mammary gland development. But its other functions were not completely understood. Much less is known about the biological actions of hPL during pregnancy, the mechanisms by which hPL exerts its biological actions, and the factors involved in the regulation of the synthesis and secretion of hPL. In our experiments, we tried to produce large amount of recombinant hPL in E. coli for further biological studies and clinical use. Several factors, like promoter, host strain, temperature, concentration of inducer and post-induction time, which could influence the expression were taken into account. Four kinds of expression vectors containing the hPL cDNA were transformed into several kinds of suitable host strains and grown at 37℃ and 30℃. The yield of recombinant hPL in different conditions was determined by SDS-PAGE. Among these combinations, pQE30-PL M15[pREP4] expressed the largest amount of recombinant hPL at 37℃, and the expressed recombinant hPL were accumulated in inclusion bodies. Expressions of the recombinant hPL in soluble form as well as pQE30-PL M15[pREP4] by changing the conditions of host strains, temperature and concentration of inducer were also performed. Although smaller amount of recombinant hPL was expressed in soluble form, relatively, greater part was still in insoluble form. The inclusion bodies were then solubilized in 8 M urea and purified by a His6 tagged affinity column under a denaturing condition. The yield of hPL was determined to be 48 mg per liter. In order to obtain hPL in the active form, the denatured protein was refolded and detected by SDS-PAGE in non-reducing conditions. Intrachain disulfide bonds could be formed either by the refolding buffer or air oxidation even in the presence of urea. And the biological activity assay was tested by the fact that hPL could stimulate erythroid maturation in K-562 cells in the presence of erythropoietin. The assay was confirmed formation of hemoglobin.