Fusion protein system designed to provide color to aid in the expression and purification of proteins in Escherichia coli.
We have designed and constructed a new fusion expression vector (pKW32), which contains the His-tagged Vitreoscilla hemoglobin (VHb) coding gene upstream of the multiple cloning site. The pKW32 vector was designed to express target proteins as VHb fusions, which can be purified in one step by affinity chromatography. Due to the color of the heme in VHb, the VHb-fused target proteins have a red color that provides a visual aid for estimating their expression level and solubility. The red color can also be used as a visual marker throughout purification, while the concentration of the fusion protein can be determined by measuring the amount of VHb using carbon monoxide difference spectra. In addition, because of inherently high solubility of VHb, the fusion can increase the solubility of sparingly soluble target proteins. Target proteins can be easily separated from His-tagged VHb due to the presence of a thrombin-cleavage site between them. A mutant VHb, the soluble domain of Vitreoscilla cytochrome bo subunit II, and HIV integrase expressed and purified using the pKW32 system have native function. In addition, the integrase, which is known to be difficult to purify because of low solubility, was purified simply and without solubilizing agents using our system.
Department of Biological, Chemical, and Physical Sciences, Illinois Institute of Technology, 3101 S Dearborn Chicago, Chicago, IL 60616, USA.
Kyung-Won
KW
Park
Dale A
DA
Webster
Benjamin C
BC
Stark
Andrew J
AJ
Howard
Kyung-Jin
KJ
Kim
D001483:Base Sequence; D003116:Color; D045303:Cytochromes b / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D019427:HIV Integrase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006454:Hemoglobins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D020589:Vitreoscilla / Q000235:genetics
D006454:Hemoglobins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D045303:Cytochromes b; D019427:HIV Integrase