Abstract
The cDNA sequence for the mature form of the zein protein A20 was inserted into the lac cloning region of two different M13mp phage vectors. Translation of these recombinant phage in E. coli cells produced a β-galactosidase-zein fusion protein. Another zein clone was constructed in which the entire coding sequence, including that of the signal peptide, was cloned into a M13mp vector. This clone was designed to produce a pre-zein protein which did not contain any β-galactosidase amino acids. Expression of these phage-coded proteins in E. coli cells was detected on Western blots using zein antibodies. Expression of the β-galactosidase-zein fusion protein was extremely low, comprising less than 1% of the total E. coli proteins. Levels of expression were increased slightly when this same sequence was cloned into a pUC-derived expression plasmid containing the highly efficient trp-lac promoter.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 157-175 |
| Number of pages | 19 |
| Journal | Journal of Biotechnology |
| Volume | 2 |
| Issue number | 3-4 |
| DOIs | |
| State | Published - May 1985 |
Bibliographical note
Funding Information:We thank Kris Kohn and Stephanie Young for help in preparing the manuscript, Beth Lewis for zein antiserum, and Diane Vician for help in the DNA synthesis. This work was supported by the Department of Energy DE-FG02-84ER13210 (J.M.) and NIH GM 24756 (I.R.).
Keywords
- M13mp and pUC vectors
- oligonucleotide-directed in vitro mutagenesis
- protein engineering