Highly efficient protein expression and purification using bacterial hemoglobin fusion vector.
Recently developed bacterial hemoglobin (VHb) fusion expression vector has been widely used for the production of many target proteins due to its distinctive properties of expressing fusion protein with red color which facilitates visualization of the steps in purification, and increasing solubility of the target proteins. However, after intensive use of the vector, several defects have been found. In this report, we present a modified VHb fusion vector (pPosKJ) with higher efficiency, in which most of the defects were eliminated. First, it was found that thrombin protease often digests target protein as well as inserted thrombin cleavage site, so it was replaced by a TEV cleavage site for more specific cleavage of VHb from target protein. Second, a glycine-rich linker sequence was inserted between 6x his-tag and VHb to improve the affinity of 6x his-tag to Ni-NTA resin, resulting in higher purity of eluted fusion protein. Third, EcoRI and XhoI restriction sites located elsewhere in the vector were removed to make these restriction sites available for the cloning of target protein coding genes. A pPosKJ vector was fully examined with an anti-apoptotic BCL-2 family member of Caenorhabditis elegans, CED-9. A C-terminal VHb fusion expression vector (pPosKJC) was also constructed for stable expression of target proteins that may be difficult to express with an N-terminal fusion. Vaccinia-related kinase 1 (VRK1) was also successfully expressed and purified using the vector with high yield. Taken together, we suggest that the VHb fusion vector may be well suited for high-throughput protein expression and purification.
Kwon
Soo-Young
SY
X-ray Research Group, Pohang Accelerator Laboratory, Pohang, Kyungbuk 790-784, Republic of Korea.
Choi
Yoon-Joo
YJ
Kang
Tae-Hong
TH
Lee
Kwang-Hoon
KH
Cha
Sun-Shin
SS
Kim
Gyung-Hwa
GH
Lee
Heung-Soo
HS
Kim
Kyong-Tai
KT
Kim
Kyung-Jin
KJ
D000595:Amino Acid Sequence / Q000235:genetics; D051017:Apoptosis Regulatory Proteins; D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D001483:Base Sequence / Q000235:genetics; D029742:Caenorhabditis elegans Proteins / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D003596:Cytosine / Q000737:chemistry; D005822:Genetic Vectors; D005998:Glycine / Q000737:chemistry; D006147:Guanine / Q000737:chemistry; D015801:Helminth Proteins / Q000302:isolation & purification / Q000378:metabolism; D006454:Hemoglobins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D009994:Osmolar Concentration; D011518:Proto-Oncogene Proteins / Q000302:isolation & purification / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D013696:Temperature
D051017:Apoptosis Regulatory Proteins; D001426:Bacterial Proteins; D029742:Caenorhabditis elegans Proteins; C087233:Ced-9 protein, C elegans; D015801:Helminth Proteins; D006454:Hemoglobins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; D006639:Histidine; D006147:Guanine; D003596:Cytosine; D005998:Glycine