A novel method for promoting heterologous protein expression in Escherichia coli by fusion with the HIV-1 TAT core domain.
The human immunodeficiency virus type 1 (HIV-1) transactivator of transcription (TAT) protein, a member of the protein transduction domain (PTD) superfamily, can deliver heterologous proteins across most biomembranes without losing bioactivity. However, there is no report on whether the TAT core domain containing the sequence 'YGRKKRRQRRR' has other functions. As the TAT core domain is most basic (pI=12.8) and has biomembrane crossing ability, we hypothesized it might probably influence the protein expression level due to subcellular redistribution of target proteins in the cells. To address this issue, we constructed the prokaryotic expression vector pET28b-TAT-EGFP (using the vector pET28b-EGFP for control) containing the core domain coding region, and transformed the vector into E. coli BL21 (DE3) cells for expression of the enhanced green fluorescent protein (EGFP) with the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG). Equal amount of the total proteins were fractionated using 15% SDS-PAGE and identified by western blot, and the plasmid copy number was assayed by Southern blot. In order to further study the subcellular localization of heterologous proteins in E. coli cells, the cytoplasmic and periplasmic components were extracted by chloroform and osmotic shock techniques. Interestingly, our data showed that the TAT core domain was not only able to promote the heterologous protein expression in E. coli, but also improve the yields and the solubility of heterologous proteins, while the plasmid copy number of TAT-containing clones and TAT-free clones was not affected by the TAT core domain. In addition, the TAT-tagged protein was mainly localized in the cytoplasm and also accumulated in the periplasmic space along with the time for protein expression, while in contrast, the TAT-free protein was mainly expressed in the periplasm and only a few in cytoplasm. A further examination on the distribution of the expressed proteins in cytoplasm and periplasm suggested that the TAT core domain might promote protein expression in the cytoplasm initially and then partially deliver them across the cytomembrane to the periplasmic space in a concentration-dependent manner. Taken together, our current data have provided a novel method for improving heterologous protein expression in prokaryotic cells by fusion with the TAT core domain, which will promote expression efficiency of bioactive proteins for protein engineering.
Wu
Yonghong
Y
State Key Laboratory of Proteomics, Beijing Institute of Radiation Medicine, Taiping Road 27, 100850, Beijing, China.
Ren
Changhong
C
Gao
Yan
Y
Hou
Bing
B
Chen
Tingfang
T
Zhang
Chenggang
C
D019677:Artificial Gene Fusion / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D054322:tat Gene Products, Human Immunodeficiency Virus; D049452:Green Fluorescent Proteins