Anti-freeze proteins (AFP), produced by fish, plants and beetles, has a widespread application potential within the food- and oil industry, among others. The complications and costliness of large scale-production hinder research in the application of AFP. Until now, Escherichia coli (E. coli) has been the production bacterium of RmAFP#1. This project explores the possibility of using Brevibacillus choshinensis SP3 (B. choshinensis) as production bacterium for RmAFP#1. Unlike E. coli BL21, used as control in this project, whose vector has a lac-operator, the vector of B. choshinensis is a constitutive promoter, not needing to be induced. Furthermore, it has a signal peptide that cause secretion of the protein complex to the growth medium. This prevents intracellular accumulation, and increases the maximum production per bacterium. The vector also holds the gene for green flourescent protein (GFP), and a his-tag. The GFP is easier to detect than AFP, and the his-tag makes purification with a nickel column possible. The protein complex is: signal peptide-GFP-RmAFP#1-histag. This project shows that the used strain of B. choshinensis expresses the protein complex and secretes it to the growth medium. B. choshinensis expressed a larger amount of the protein complex than E. coli. The growth temperatures for both bacteria were 250C, 280C and 370C. The largest production obtained was at 280C for B. choshinensis after 32 hours and at 370 C for E. coli, after 3 hours. AFP starts irreversible denaturation at 280C. Since B. choshinensis produce more RmAFP#1 than E. coli and at a more favorable temperature for RmAFP#1 it is worth to continue working with B. choshinensis.
|Educations||Molecular Biology, (Bachelor/Graduate Programme) Undergraduate or graduate|
|Publication date||26 Jun 2014|