title: Engineering of a wheat germ expression system to provide compatibility with a high throughput pET-based cloning platform.
abstract: Wheat germ cell-free methods provide an important approach for the production of eukaryotic proteins. We have developed a protein expression vector for the TNT((R)) SP6 High-Yield Wheat Germ Cell-Free (TNT WGCF) expression system (Promega) that is also compatible with our T7-based Escherichia coli intracellular expression vector pET15_NESG. This allows cloning of the same PCR product into either one of several pET_NESG vectors and this modified WGCF vector (pWGHisAmp) by In-Fusion LIC cloning (Zhu et al. in Biotechniques 43:354-359, 2007). Integration of these two vector systems allowed us to explore the efficacy of the TNT WGCF system by comparing the expression and solubility characteristics of 59 human protein constructs in both WGCF and pET15_NESG E. coli intracellular expression. While only 30% of these human proteins could be produced in soluble form using the pET15_NESG based system, some 70% could be produced in soluble form using the TNT WGCF system. This high success rate underscores the importance of eukaryotic expression host systems like the TNT WGCF system for eukaryotic protein production in a structural genomics sample production pipeline. To further demonstrate the value of this WGCF system in producing protein suitable for structural studies, we scaled up, purified, and analyzed by 2D NMR two (15)N-, (13)C-enriched human proteins. The results of this study indicate that the TNT WGCF system is a successful salvage pathway for producing samples of difficult-to-express small human proteins for NMR studies, providing an important complementary pathway for eukaryotic sample production in the NESG NMR structure production pipeline.
authors.affiliation: Center for Advanced Biotechnology and Medicine, University of Medicine and Dentistry of New Jersey, Piscataway, 08854, USA.
authors.forename: Li
authors.initials: L
authors.lastname: Zhao
authors.forename: Kate Q
authors.initials: KQ
authors.lastname: Zhao
authors.forename: Robin
authors.initials: R
authors.lastname: Hurst
authors.forename: Michael R
authors.initials: MR
authors.lastname: Slater
authors.forename: Thomas B
authors.initials: TB
authors.lastname: Acton
authors.forename: G V T
authors.initials: GV
authors.lastname: Swapna
authors.forename: Ritu
authors.initials: R
authors.lastname: Shastry
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Kornhaber
authors.forename: Gaetano T
authors.initials: GT
authors.lastname: Montelione
mesh_terms: D002474:Cell-Free System; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D015202:Protein Engineering; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D011994:Recombinant Proteins / Q000096:biosynthesis*
chemical_list: D011506:Proteins; D011994:Recombinant Proteins