Part:BBa_K942004:Experience

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Applications of BBa_K942004

Our shuttle sequence can be quite useful to other iGEM teams and anyone with the objective of producing or comparing the expression of proteins in two different expression systems. Coupled with an codon optimization for Pichia p. and a strain capable of reading rare codons like Rosetta Gami DE3 pLys, Bl21 star, the outcome can be a simultanious expression in both organisms coming from one single genetic sequence.

User Reviews

UNIQ0f1e73d6b7cae58b-partinfo-00000000-QINU UNIQ0f1e73d6b7cae58b-partinfo-00000001-QINU

This construct was used for the expression of Der f 2 in Pichia pastoris. Also, thanks to its B.P.S. (bacterial promoter sequence) it is intended to be expressed in Escherichia coli.

With our current evidence, and considering the fact that Der f 2 had been previously proposed to have antibacterial activity (Ichikawa S, 1998), we believe it is possible that BL21(DE3) and JM109(DE3) had been effectively transformed with the plasmid; but due to leaky expression of Der f 2, the cells might have been unable to grow any further and produce colonies. Rosetta gami pLysS, on the other hand, has the capacity to suppress expression from the T7 promoter; and this may be the reason for its survival and growth.

We achieved expression of the protein in P. pastoris cultures grown in 0.5% methanol and enough aeration. Because this part uses a secretion signal for yeasts, the protein was mainly localized in the growth medium. After being harvested, the cultures were centrifuged (5000g , 4º C, 5 min), and the supernatant was collected and purified using Vivapure Metal Chelate Maxi spin columns (Sartorius Stedim Biotech). Futhermore, the samples were concentrated with 3 kDa ultracentrifuge filters, obtaining a final concentration of 0.37 ug/uL in 500ul.

We were able to detect the expression of this protein on a SDS-PAGE after purifying it with a His-tag affinity column. Currently, we are trying different conditions to achieve the expression in BL21 Star.

Tricine 16% gels were usedn order to see the protein by sds-page. Third lane = Derf (16KDa molecular weight) Fourth Lane = SeeBlue® Plus2 Pre-Stained