Continuous protein production device and method
A production device and production method technology, which is applied in the field of continuous protein production devices, can solve the problems of affecting protein yield, expensive reaction reagents and reaction containers, etc., and achieve the effect of expanding reaction volume, flexible reaction and expanding system
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Embodiment 1
[0036] refer to figure 1 , a protein continuous production device provided by an embodiment of the present invention includes a material exchange unit 3, an aeration unit 5, a liquid supply unit, a reaction temperature control unit 9, and a gas source 10; wherein, the material exchange unit 3 includes a pore size of 10KDa semi-permeable membrane (cellulose lipid membrane) 2 separated reaction chamber 31 and supply chamber 32, the reaction chamber 31 is at least used to accommodate the liquid phase cell-free protein synthesis reaction system, and the supply chamber 32 is at least used to accommodate the liquid phase supplement system, the supply chamber 32 cooperates with the supply unit of the liquid phase replenishment system to form a working circuit for the circulation of the liquid phase replenishment system.
[0037] The ventilation unit 5 includes a liquid delivery chamber 51 and a gas delivery chamber 52 separated by a partition mechanism 4 formed of a gas-permeable hyd...
Embodiment 2
[0040] Example 2: Expression of human urinary trypsin inhibitor (hUTI) with fluorescent protein (sfGFP)
[0041] As an example of the continuous protein production method, the continuous exchange method of the protein continuous production apparatus shown in Example 1 was implemented, and protein synthesis was performed using the Escherichia coli extract.
[0042] 1. Expression vector
[0043] In the present invention, the sfGFP-hUTI gene expression vector is used for cell-free protein production. The sfGFP-rhUTI expression vector was used to confirm the feasibility of applying the present invention to protein production from cell-free.
[0044] The expression vector construction process is as follows:
[0045] (1) Search the cDNA of human urinary trypsin inhibitor in GeneBank, add a histidine tag and sfGFP base to the N-terminus, and perform gene synthesis on the above gene. See SEQ ID NO: 1 for the gene synthesis sequence.
[0046] (2) Using pIJ8660F: GGTGACGAAGAACTGCTGta...
Embodiment 3
[0064]Example 3 Expression of human urinary trypsin inhibitor (hUTI) with fluorescent protein (sfGFP) (the lower limit of the parameter range of the cell-free reaction system)
[0065] Expression vector construction, cell lysate preparation, and continuous exchange cell-free protein expression sfGFP-hUTI protein expression and purification experimental methods are as described in Example 2, and the concentrations of the following components are changed:
[0066] Preparation of the solution of the reaction system
[0067] The expression system components are as follows: 5ng / μL pIJ8660-sfGFP-hUTI, 2mM phosphate buffer, 0.5mM ATP, 0.5mM UTP, 0.5mM CTP, 0.5mM GTP, 50mM potassium glutamate, 0.5mM magnesium glutamate, 0.5 mM potassium oxalate, 0.5 mM amino acid mixture, Escherichia coli extract with a volume ratio of 15%.
[0068] Preparation of Supplementary System Solutions
[0069] The composition of the supplement solution is as follows: 2mM phosphate buffer, 0.5mM ATP, 0.5mM ...
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