Study On The Recombinant VHb To Antagonize The Oxygen Fluctuation In E.coli Fermentation And Its Auxiliary Expression Of RhARG

In order to solve the problem of oxygen limited in the production of protein genetically engineered products,we designed the self-assembly peptide sequence linked to the C-terminus of Vitroeoscilla hemoglobin by molecular biotechnology,constructing self-assembled recombinant hemoglobin proteins VHb9 a and VHb22 a.With the expectation for obtaining a new method and strategy to modify functional proteins,we investigated and compared physiological functions of VHb9 a and VHb22 a with VHb in the soluble expression of GFPuv and rh ARG.First,the recombinant protein was purified and analyzed by spectroscopy.The results showed that the Vitroeoscilla hemoglobin linked with self-assembled peptide sequence could form plasma membrane bounding aggregates.The intermolecular ?-fold structure and the secondary structure and spatial structure of the porphyrin protein monomer in the aggregates did not significantly change.It was deduced that the plasma membrane binding property of the recombinant porphyrin proteins linked with self-assembled peptide sequence will regulate the physiological functions of oxygen metabolism in the genetically engineered fermentation host cells.Secondly,the co-expression vectors of porphyrin proteins and GFPuv was constructed to study the ability of self-assembly protein expressing GFPuv without cytotoxicity.The results showed that the porphyrin protein linked with self-assembled peptide sequence had a higher ability of supporting exogenous protein expression.VHb9 a self-assembled protein could increase the expression of GFPuv by 53%,and its effect was better than 25% of wild-type VHb and 34% of VHb22 a.The VHb22 A expressing bacteria had a higher survival rates at higher H2O2 concentrations.The self-assembled VHb had a higher oxygen uptake rate and higher oxygen storage capacity as well,compared with the control bacteria.All results indicated that the plasma membrane binding properties confer a new oxygen-related biological activity on the functional protein VHb.Furthermore,in order to explore the soluble expression ability of porphyrin protein assisted cytotoxic protein rh ARG,the co-expression vectors of porphyrin protein and rh ARG were constructed,the permeabilization method of screening rh ARG expression by a large-scale and high-throughput was established,the conditions to express rh ARG were optimized and analyzed.The results showed that the rh ARG activity of VHb22a-assisted host cells was the best in the case of relatively high oxygen and oxygen deficiency,and increasing the rh ARG activity was significantly by 15% and 31% compared with the negative control group.In the production of rh ARG in 5L fermentor with 4L medium,the host bacteria of VHb22 a were 62% higher in rh ARG activity.Finally,in order to study the positional relationship of self-assembled VHb in the respiratory chain,the genes of terminal oxidase d and terminal oxidase o in Escherichia coli host cells were selectively knocked out by gene knockout technique and the structure and functional location of recombinant porphyrin proteins on cell membrane were judged by the growth of bacteria.The results showed that the selfassembled VHb reduced the biological activity of the protein related to low-oxygen respiratory chain other than the intracellular cytochrome oxidase d,and this effect of VHb22 a on the respiratory chain was higher than that of VHb9 a in the case of trifle scarcity of oxygen.When the oxygen supply,however,was seriously insufficient,VHb22 a can perform its physiological function as like cytochromed.