Human tyrosinase expression carrier and its use
A technology of tyrosinase and expression vector, which is applied in the field of human tyrosinase expression vector and its application, can solve the problems of inability to glycosylate, glycosylation or insufficient glycosylation, and easy formation of insoluble inclusion bodies, etc. Achieve the effect of lower production cost and easy construction
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Embodiment 1
[0032] Example 1, Construction of human tyrosinase Pichia pastoris expression vector pPIC3.5KHis-tyr
[0033] Such as figure 1 As shown, the construction process of the vector pPIC3.5KHis-tyr includes the following steps:
[0034] 1. Cloning of the gene encoding the His-tag and construction of the recombinant vector pPIC3.5Khis containing the gene
[0035] The primer sequences are as follows:
[0036] Primer 1: (upstream primer) 5'-gaagtacgtaatgggcagcagccatcatcatcatc-3'
[0037] Primer 2: (downstream primer) 5'-gagagaattcaccccatttgctgtccaccagt-3'
[0038] 1. Using the plasmid pET-28a(+) as a template, under the guidance of primers 1 and 2, perform PCR amplification of the His-tag gene. The PCR reaction conditions are: 94°C for 3 minutes; 94°C for 1 minute, 57°C for 1 minute minutes, 30 seconds at 72°C, 30 cycles; extension at 72°C for 10 minutes, and incubation at 4°C. The PCR product was detected by 1% agarose gel electrophoresis, which showed that a DNA amplification fr...
Embodiment 2
[0046] Example 2. Induced expression and Western-blot identification of human tyrosinase
[0047] 1. Induced expression of human tyrosinase
[0048] 1. Linearization of plasmid pPIC3.5KHis-tyr
[0049] The human tyrosinase Pichia expression vector pPIC3.5KHis-tyr obtained in Example 1 was digested with SacI restriction endonuclease, and the electrophoresis-pure linearized plasmid pPIC3.5KHis was recovered after 1% agarose gel electrophoresis -tyr.
[0050] 2. Transform the linearized plasmid pPIC3.5KHis-tyr into Pichia pastoris
[0051] The specific process includes the following steps:
[0052] 1) Take Pichia pastoris GS115 stored in a glycerol tube, streak it on a YPD agar plate, culture it at 30°C for 12-24 hours to activate it, pick a single colony and inoculate it in YPD liquid medium, and culture it at 30°C until OD 600 The value is 1.4-1.5.
[0053] 2) Pipette 1 mL of the culture solution from step 1) into EP centrifuge tubes (two tubes in parallel), centrifuge at...
Embodiment 3
[0069] Embodiment 3, the purification of human tyrosinase protein
[0070] Loading buffer (MCAC-0): Tris-HCl 20 mM pH7.9, NaCl 500 mM, glycerol 10%, PMSF (phenylmethanesulfonyl fluoride) 1 mM.
[0071] Elution buffer (MCAC-500): imidazole 500 mM, Tris-HCl 20 mM, NaCl 500 mM, glycerol 10%, PMSF 1 mM.
[0072] Purify the target protein expressed in Example 2 with nickel ion affinity column chromatography, the specific steps are as follows:
[0073] 1) Collect the yeast cells induced to express in Example 2, dissolve them in loading buffer MCAC-0, homogenize at low temperature and centrifuge at 15,000 rpm for 10 min, and collect the crude protein solution;
[0074] 2) Add the crude protein solution to the upper layer of a nickel ion affinity chromatography column (purchased from QIAGEN), and maintain a flow rate of 0.01ml / sec until the target protein is completely adsorbed;
[0075] 3) Add elution buffer MCAC-500 for elution, collect 5ml protein eluate to obtain purified human ...
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