The invention provides a multi-copy
metallothionein recombinant expression vector and a method thereof for high-efficiency expression of
metallothionein, and belongs to the technical field of biological
chemistry. The method is as follows: constructing the multi-copy
metallothionein recombinant expression vector; converting the constructed multi-copy metallothionein
recombinant expression vector in
pichia pastoris GS115 or KM71, and screening on a
histidine-free culture medium to obtain recombinant
bacteria; inoculating the screened recombinant
bacteria in a BMGY culture solution, rapidly oscillating for culture, centrifugally collecting
bacteria, resuspending with the culture solution, diluting, rapidly oscillating for culture until log phase, adding
methanol, and feeding the
methanol in batches to continue to cultivate; collecting the bacteria, ultrasonically crushing, collecting supernatant by
centrifugation, and separating and purifying the supernatant to obtain the metallothionein. The method overcomes the defects of a traditional metallothionein preparation method, can effectively avoid the waste of a lot of animal resources and bacteria sourced
endotoxin contamination, reduces the production cost, and improves the
protein yield.