Construction method for more stable mutant of humanized proteasome activity factor
A technology of proteasome and activator, applied in the field of molecular biology and structural biology, can solve the problem of inability to obtain proteasome activator
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Embodiment 1
[0053] Construction of human proteasome activator mutant REGgammaLinker independent of ubiquitin and ATP
[0054] 1. Molecular cloning of REGgammaLinker containing proteasome activating factor mutant
[0055] 1) Using the nucleotide sequence of the REGgamma parent protein as a template, use the two pairs of primers (SEQ ID No.2 to SEQ ID No.5) of FP1 / linker2 and RP1 / linker1 to perform PCR reactions to amplify the front of the replacement site The end segment and the end segment behind. The reaction system is as follows:
[0056]
[0057] 2) PCR reaction program
[0058]
[0059] 2. Recovery of Mutant REGgammaLinker Gene Sequence PCR Product
[0060] After the PCR, the product was electrophoresed on a 1% agarose gel and cut to recover a target fragment of about 600 bp. Specific steps are as follows:
[0061] 1) After agarose gel electrophoresis, observe the gel running results under ultraviolet light, cut out the target fragment and put it into a 1.5ml sterilized EP...
Embodiment 2
[0100] Prokaryotic expression and purification of mutant protein REGgammaLinker, crystal screening and structural analysis;
[0101] 1. Transform E.coli BL21 with the recombinant plasmid carrying the full-length gene of the REGgammaLinker mutant
[0102] The transformation step is the same as the transformation step of the ligated product into E.coli trans5α in step 6, except that after adding LB medium to the system, the shaking culture time at 37°C is 1h. The bacterial solution was spread on a plate containing 100mg / l Ampicillin antibiotic, and cultured upside down at 37°C for 12h.
[0103] 2. IPTG induces heterologous expression of REGgammaLinker mutant proteins
[0104] a. Pick a single colony containing the recombinant plasmid on the plate and inoculate it into 5 ml of high-temperature sterilized LB liquid medium supplemented with 100 mg / l Ampicillin antibiotic, and cultivate overnight at 37°C with shaking;
[0105] b. Transfer to liquid LB medium supplemented with 10...
Embodiment 3
[0153] Activation of proteasome by REGgamma native and REGgammaLinker mutants
[0154]For the method of enzyme activity experiment, please refer to the article "Lysine 188 substitutions convert the pattern of proteasome activation by REGγto that of REGs αandβ" published by JunLi, Martin Rechsteiner et al. in the EMBO Journal in 2001. method with slight improvements.
[0155] The specific experimental steps are as follows:
[0156] a. Add 40ul assay buffer (50mM Tris PH7.5, 25mMKCl 10mMNaCl1MgCl 2 ).
[0157] b. Add 30ul assaybuffer, 10ul proteasome (10ug / ml) to the negative control;
[0158] c. Add 20ul assay buffer, 10ul REGα (100ug / ml) and 10ul proteasome to the positive control;
[0159] d. Add 20ul assay buffer, 10ul REGgamma Native (100ug / ml) and 10ul proteasome to sample No. 1 group;
[0160] e. Add 20ul assay buffer, 10ul REGgammaLinker mutant (100ug / ml) and 10ul proteasome to sample No. 2 group
[0161] f. Mix the components of the appeal at 30 degrees for 10 min...
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