Accession Number:

ADA282411

Title:

Expression and Partial Purification of Several Truncated Forms of Chloroplast Translational Initiation Factor 2 from Euglena Gracilis

Descriptive Note:

Master's thesis

Corporate Author:

AIR FORCE INST OF TECH WRIGHT-PATTERSONAFB OH

Personal Author(s):

Report Date:

1994-01-01

Pagination or Media Count:

74.0

Abstract:

Two truncated forms of Euglena gracilis chloroplast initiation factor 2 have been engineered at the DNA level and expressed at a high level in Escherichia coli. The DNA was obtained by PCR amplification of a cDNA encoding a portion of IF-2chl. The PCR primers were custom-designed and added a restriction site at each end for sub-cloning into the QIAexpress expression system. This expression system provides excellent protein yield in E. coli and adds six histidine residues which bind Ni-NTA affinity resin. Two polypeptides were produced. A 30 kDa polypeptide which contains the highly homologous guanine nucleotide binding region of IF-2chl was designed and has been designated the G-domain. The second protein, designated IF-2chl gamma, has been designed to be approximately the same size 66 kDa as E. coli IF-2 gamma. Based on sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis, both proteins have been expressed in E. coli, and production of the G-domain was superb. IF-2chl gamma was present at a much lower relative concentration and its presence greatly retarded cell growth. The G-domain protein was also purified under native conditions to approximately 85-90 purity. A four step process utilizing affinity chromatography and high-pressure liquid chromatography was followed, and the presence of the protein was monitored by SDS-PAGE. Author

Subject Categories:

  • Biochemistry

Distribution Statement:

APPROVED FOR PUBLIC RELEASE