Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.358182
Title: Expression, characterisation and purification of complement Cls domain-1
Author: McAlister, Mark S. B.
Awarding Body: University of Aberdeen
Current Institution: University of Aberdeen
Date of Award: 1993
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Abstract:
The aim of this project was to develop an expression system and protein purification system to allow production of the N-terminal domain of complement Cls (Cls-I) for structural studies by NMR spectroscopy or X-ray crystallography, and possibly for functional studies. A yeast expression/secretion vector has been constructed by ligating PCR-isolated Cls-I to the yeast α-factor leader sequence and the yeast phosphoglycerate kinase expression cassette. Yeast cells transformed with the expression vector produced two bands on SDS-PAGE that differed by 1-2kDa in apparent molecular weight (14kDa and 12kDa). The 14kDa (a.Mr.) form of Cls-I was N-terminally intact and mass spectrometry indicated that it had the expected mass of C-terminally intact unmodified Cls-I. The 12kDa (a.Mr.) form of Cls-I was N-terminally recessed by two amino-acids, and mass spectrometry suggested that it was also C-terminally intact and not further post-translationally modified. Glycoprotein detection blotting suggested that Cls-I was not glycosylated and a dephosphorylation experiment suggested that Cls-I was not phosphorylated in agreement with the mass spectrometry results. The N-terminal cleavage of Cls-I appeared to be mediated by the STE13 encoded diaminopeptidase A, since 60% of Cls-I secreted from a ste13 partially deficient mutant strain (ER109-5C) was N-terminally intact, but only 25-40% of Cls-I secreted from the `wild-type' BJ1991 transformants was intact. SDS-PAGE of intracellular protein suggested that there was not a significant intracellular accumulation of Cls-I. As yeast transformant cultures entered late transition/stationary phase (O.D.600nm = 3.5-4.5) a C-terminal proteolytic cleavage of both 14kDa and 12kDa (a.Mr.) forms of Cls-I appeared to occur, resulting in the formation of doublets of both bands. This was avoided by harvesting the cultures at O.D.600nm = 3-3.5.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.358182  DOI: Not available
Keywords: Immunology Molecular biology Cytology Genetics Biochemistry Microbiology
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