Expression and purification of human placenta lactogen in Escherichia coli.
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 quantities 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 degrees 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 degrees C. However, the expressed recombinant hPL was accumulated in inclusion body forms. The inclusion bodies were solubilized in 8M urea and purified by a His6 tagged affinity column under denaturing condition and the final yield of hPL was determined to be 48 mg/L. Intra-chain disulfide bonds could be formed either by oxidation in the refolding buffer or by air oxidation in the presence of urea. The biological activity was examined by the fact that hPL could stimulate erythroid 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.
Lan
Pei Ching
PC
Department of Biological Science and Technology, National Chiao Tung University, 75 Po-Ai Street, Hsinchu, Taiwan, ROC.
Tseng
Chi Feng
CF
Lin
Meng Chia
MC
Chang
C Allen
CA
D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli; D005260:Female; D015870:Gene Expression; D006801:Humans; D020014:K562 Cells; D010928:Placental Lactogen / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*
D011993:Recombinant Fusion Proteins; D010928:Placental Lactogen