Advanced

An in vivo system for measuring solubility of proteins without activity in Escherichia coli

Cicek, Hasan (2012) KEMT30 20111
Department of Chemistry
Abstract
Protein design can be used to make better variants of a protein by changing the primary structure of proteins. An assay is needed to select for a better protein variant, generally these assays work on proteins with an activity. But sometimes proteins are designed to obtain a specific structure instead of an activity or function. These proteins often have no activity and there is no easy way to select for this kind of proteins at the present time. Formation of the correct protein structure is not easily assayed; however solubility is both a good indicator of a stable protein and of a folded protein. In this thesis a high-through-put method that measure solubility that is intended to work with proteins without function was developed. Instead... (More)
Protein design can be used to make better variants of a protein by changing the primary structure of proteins. An assay is needed to select for a better protein variant, generally these assays work on proteins with an activity. But sometimes proteins are designed to obtain a specific structure instead of an activity or function. These proteins often have no activity and there is no easy way to select for this kind of proteins at the present time. Formation of the correct protein structure is not easily assayed; however solubility is both a good indicator of a stable protein and of a folded protein. In this thesis a high-through-put method that measure solubility that is intended to work with proteins without function was developed. Instead of looking at the protein of interest, the attention is turned to the response of Escherichia coli that produces the proteins. Previous research has shown that E. coli induces a heat-shock response when unfolded proteins are over-expressed. By using this phenomena and measuring the over-expression of a protein of interest and at the same time measure the induction of a heat-shock protein promoter, a sensitive system that select for proteins with higher solubility can be obtained. The heat shock promoter and the over-expression of the protein of interest were produced on separate vectors, and were then co-expressed in one plasmid using ligation independent cloning (LIC) .To test if our developed system works as intended, two co-expression vectors were designed. One of the co-expression vectors over-express a variant of the lambda repressor (N102LT) which is folded at 37 °C and the other co-expressed vector over-express a variant of N102LT which has a single mutation(LA57) which renders the protein unfolded at 37 °C. The co-expressed vector with LA57 construct was cloned into the vector, while the construct with N102LT was not obtained. Both constructs are needed to evaluate our system. Fluorescence proteins are used as reporters, and to evaluate the strength of the fluorescence signal from ECFP in a cell culture, ECFP was expressed by itself and a strong signal was obtained. This indicates that fluorescence measurement can be up scaled to fit 96-microtitre plates to make it high-through-pu (Less)
Please use this url to cite or link to this publication:
author
Cicek, Hasan
supervisor
organization
course
KEMT30 20111
year
type
H2 - Master's Degree (Two Years)
subject
keywords
Proteinvetenskap
language
English
id
2861363
date added to LUP
2012-07-06 15:16:33
date last changed
2015-10-01 12:20:12
@misc{2861363,
  abstract     = {Protein design can be used to make better variants of a protein by changing the primary structure of proteins. An assay is needed to select for a better protein variant, generally these assays work on proteins with an activity. But sometimes proteins are designed to obtain a specific structure instead of an activity or function. These proteins often have no activity and there is no easy way to select for this kind of proteins at the present time. Formation of the correct protein structure is not easily assayed; however solubility is both a good indicator of a stable protein and of a folded protein. In this thesis a high-through-put method that measure solubility that is intended to work with proteins without function was developed. Instead of looking at the protein of interest, the attention is turned to the response of Escherichia coli that produces the proteins. Previous research has shown that E. coli induces a heat-shock response when unfolded proteins are over-expressed. By using this phenomena and measuring the over-expression of a protein of interest and at the same time measure the induction of a heat-shock protein promoter, a sensitive system that select for proteins with higher solubility can be obtained. The heat shock promoter and the over-expression of the protein of interest were produced on separate vectors, and were then co-expressed in one plasmid using ligation independent cloning (LIC) .To test if our developed system works as intended, two co-expression vectors were designed. One of the co-expression vectors over-express a variant of the lambda repressor (N102LT) which is folded at 37 °C and the other co-expressed vector over-express a variant of N102LT which has a single mutation(LA57) which renders the protein unfolded at 37 °C. The co-expressed vector with LA57 construct was cloned into the vector, while the construct with N102LT was not obtained. Both constructs are needed to evaluate our system. Fluorescence proteins are used as reporters, and to evaluate the strength of the fluorescence signal from ECFP in a cell culture, ECFP was expressed by itself and a strong signal was obtained. This indicates that fluorescence measurement can be up scaled to fit 96-microtitre plates to make it high-through-pu},
  author       = {Cicek, Hasan},
  keyword      = {Proteinvetenskap},
  language     = {eng},
  note         = {Student Paper},
  title        = {An in vivo system for measuring solubility of proteins without activity in Escherichia coli},
  year         = {2012},
}