Expression and Partial Purification of Several Truncated Forms of Chloroplast Translational Initiation Factor 2 from Euglena Gracilis
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-2(chl). 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-2(chl) was designed and has been designated the G-domain. The second protein, designated IF-2(chl) 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-2(chl) 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)
Document Details
- Document Type
- Technical Report
- Publication Date
- Jan 01, 1994
- Accession Number
- ADA282411
Entities
People
- Kevin E. Blanchard
Organizations
- Air Force Institute of Technology