short highly charged poly-amino acids for difficult protein production

Santema, Lars (2021) short highly charged poly-amino acids for difficult protein production. Master's Research Project 2, Biomolecular Sciences.

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Abstract

In this study it was found that the addition of a solubility enhanced protein tag will improve the overall expression of recombinant proteins in E.coli. The tag consists of six till nine highly charged arginines or aspartic acids to change the overall isoelectric point of the protein. The arginine tag seems to improve the yield the most in small oxygen poor conditions and the aspartic acid tag in oxygen richer cultures. The mode of action of the tag is unknown. Using this technique the expression of gamete fusion associated sperm protein Spaca6 is enhanced for the creation of a mouse antibody. The yield of ARMS2 and its Age related macular degeneration related mutated form ARMS2 A69S are improved a hundred fold to study its protein chemistry. The protein chemistry of both is really similar but it appears that ARMS2 A69S degenerates quicker. Finally, the expression of the nanobody NbALFA was improved a thousand fold and the stable fluorophore td Tomato was added. The created nanobody proved to be highly specific with a high resolution. Because of the solubility enhanced protein tag the nanobody can be expressed in E.coli which is cheaper and easier. 1 litre of culture creates enough fluorophore tagged nanobody for six hundred immunostaining experiments.

Item Type:	Thesis (Master's Research Project 2)
Supervisor name:	Kortholt, A.
Degree programme:	Biomolecular Sciences
Thesis type:	Master's Research Project 2
Language:	English
Date Deposited:	05 Feb 2021 14:02
Last Modified:	05 Feb 2021 14:02
URI:	https://fse.studenttheses.ub.rug.nl/id/eprint/23925

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