Vitreoscilla Hemoglobin Functional As A Visable Label Used In Protein Purification Process

In recent years, with the rapid development of biotechnology, the development of recombinant protein technology is also very mature. In the study of protein structure and protein function and interaction, we often need high purity protein, correctly folding protein, so purifying high concentration of the target protein has become the focus of research by scientists. Fusion tags technology is a newly developed kind of recombinant DNA technology, it is mainly in the target protein gene sequence of N-terminal or C-terminal of the insertion of fusion tags encoding gene sequences, constructing fusion genome sequence. Then the fusion genome sequence is converved into a suitable host cell in vivo, expressing with the fusion protein with fusion tag. The fusion tag is defined as a segment of amino acid sequence with high affinity to a specific group or chemical group. There are two main types, one is the amino acid sequence of the small molecular weight, which can be combined with solid medium, such as His tag, the FLAG tag; one is protein tag, which can be combined with small molecule ligand, such as GST tag, FMN tag. In the experiment, we find fusion tag can increase the yield of protein, protein solubility and promote protein folding, etc., so with the fusion tag, the fusion protein will increase the corresponding yield, and solubility, and protein molecule is easy to fold. Recent studies have suggested that a protein tag with target protein form fusion protein, the fusion protein can be with color, which makes no color of the target protein be color, in the follow-up purification of the target protein it can easily be tracked and detected.Vitreoscilla hemoglobin（VHb） is a small molecular weight protein, 15.7KDa, it has high solubility, both in prokaryotes and in yeast expression, it can increase the cell concentration and growth rate, and protein yield. Alkaline Pectate Lyase（CD）, it’s molecular weight is probably 22 k D, it is a colorless protein and has alkaline pectate lyase activity. In the study of CD, it’s colorless cause it difficult to express, purify and protein content is very low. In order to overcome this problem. We introduce VHb as a fusion protein tag and fusion with CD. This fusion protein will take red, the colorless CD will also be red, in subsequent purification process it will be very easy to follow and detect. Due to the small molecular weight of VHb and high solubility, it has great help for improving CD solubility and protein yield.In the integration of VHb and CD, firstly we design the vgb gene, in the downstream with a Gly linker and thrombin cleavage site, and go to polymerase chain reaction（PCR）, the PCR reaction product is connected to the p UC19 vector with the VHb anaerobic start. Secondly, we design primers of the CD gene, in the downstream with the His tag and go to polymerase chain reaction（PCR）, the product is connected to the last constructed vector. Finally, we get the recombinant plasmid p UC19+ promoter+vgb+（Gly） 5+Thrombin+CD.The obtained p UC19+promoter+vgb+（Gly）₅+Thrombin+CD recombinant plasmid was transformed into BL21 E. coli（DE3） for expression and purification. In recombinant plasmid the anaerobic promoter can activate the expression of the fusion protein.in the program of the fusion protein expression,the expression system is at 37℃, 10 h, makes a large number of the bacteria amplification. At 20℃, 100 rpm, low temperature anaerobic induce protein expression for 30 h, the fusion protein expression is in a large number. In this process does not need to measure the OD value, also need not be induced by IPTG. Finally, using a large capacity centrifuge to centrifuge, 4000 rpm, 40 min, to collect bacteria for the downstream experiment. At the same time, the VHb and CD cells were respectively collected for the control experiment of the downstream experiment.In the process of purification of the fusion protein, it is found that the supernatant of the fusion protein is red, and it is easy to track and detect the position of the target protein when the target protein is eluted. The obtained high purity of fusion protein was respectively detected by UV, activity detection, round second chromatography and the detection of fluorescence spectrum. Compared with activity detection, round second chromatography, fluorescence spectrum of single CD and VHb.The study found that in the 4L culture system, the expression of CD in fusion protein reached about 60 mg, compared to the culture system in 4L, protein CD expression of 30mg-40 mg, protein expression increased by more than 50%. In the fusion protein, VHb and CD were active, but the activity decreased by 10% compared to VHb and CD respectively. Fluorescence analysis suggests that fusion protein and CD has similar fluorescence properties, indicating that the visual tag VHb has little impact in CD protein structure of fusion protein. there are still some differences in fusion protein fluorescence absorption compared with separate CD, may due to VHb change the the exposure degree of some amino acids on CD surface. VHb is a typical alpha helix structure, there are two negative peaks in 209 nm and 221 nm.CD is a typical beta folding and alpha helix mixing structure, CD has a negative peak at 213 nm. The Circular two chromatography of fusion protein is between the two but more tend to alpha helix, which shows that the structure of the fusion protein is not the simple superposition of the two proteins.