High-level production of uniformly 15N-and 13C-enriched fusion proteins in Escherichia coli

Abstract

An approach to produce 13C- and 15N-enriched proteins is described. The concept is based on intracellular production of the recombinant proteins in Escherichia coli as fusions to an IgG-binding domain, Z, derived from staphylococcal protein A. The production method provides yields of 40-200 mg/l of isotope-enriched fusion proteins in defined minimal media. In addition, the Z fusion partner facilitates the first purification step by IgG affinity chromatography. The production system is applied to isotope enrichment of human insulin-like growth factor II (IGF-II), bovine pancreatic trypsin inhibitor (BPTI), and Z itself. High levels of protein production are achieved in shaker flasks using totally defined minimal medium supplemented with 13C(6)-glucose and (15NH4)2SO4 as the only carbon and nitrogen sources. Growth conditions were optimized to obtain high protein production levels and high levels of isotope incorporation, while minimizing 13C(6)-glucose usage. Incorporation levels of 13C and/or 15N isotopes in purifies IGF-II, BPTI, and Z were confirmed using mass spectrometry and NMR spectroscopy. More than 99% of total isotope enrichment was obtained using a defined isotope-enriched minimal medium. The optimized systems provide reliable, high-level production of isotope-enriched fusion proteins. They can be used to produce 20-40 mg/l of properly folded Z and BPTI proteins. The production system of recombinant BPTI is state-of-the-art and provides the highest known yield of native refolded BPTI.

Three‐dimensional structure of the weakly associated protein homodimer SeR13 using RDCs and paramagnetic surface mapping

“…44,45 The full-length protein was cloned into a pET21b vector, along with a S28F mutation and a short noncleavable C-terminal hexa His tag. The transformed cells were cultured at 37 C in MJ9 minimal medium 45 containing ( 15 NH 4 ) 2 SO 4 and U-13 C-glucose as the sole nitrogen and carbon sources, respectively. SeR13 was purified using an AKTAexpress FPLC apparatus with a twostep protocol consisting of HisTrap HP affinity and HiLoad 26/60 Superdex 75 gel filtration chromatography.…”

Design of an expression system for detecting folded protein domains and mapping macromolecular interactions by NMR

“…In the strategy described here, the engineered protein is synthesized as a fusion with the B1 immunoglobulin binding domain of streptococcal protein G (GB]; 56 residues) (Gronenborn et al, 1991). As is true for other N-terminal fusion proteins, such as those containing domains from glutathione S-transferase (Smith & Johnson, 1988), maltose binding protein (di Guan et al, 1988), protein A (Nilsson et al, 1985;Jansson et al, 1996), or thioredoxin (LaVallie et al, 1993), the presence of the GB1 domain results in very high expression levels of the fusion protein and limits the need to optimize expression conditions. For most fusion proteinbased expression strategies, the N-terminal domain is rather large and is removed by proteolysis prior to NMR analysis.…”

Optimized procedure to generate heavy isotope and selenomethionine-labeled proteins for structure determination using Escherichia coli-based expression systems

“…Current protocols for production of heavy isotope and Se-Met labeled proteins using E. coli expression systems are mainly based on M9 minimal medium (Doublie 1997;Fiaux et al 2004;Leiting et al 1998;Mac et al 2006;Seo et al 2009;Zhao et al 2004) leading to very poor final biomass concentrations (OD600 of 1~2) and, consequently, low yields of labeled proteins. Some improvements have been obtained by optimizing the minimal medium (Jansson et al 1996) or by using autoinduction medium for the generation of 15 N and/or 13 C or Se-Met labeled proteins (Studier 2005;Sreenath et al 2005;Tyler et al 2005). Moreover, carbon limited fed-batch processes have been developed for production of 15 N and 13 C (Ross et al 2004) or Se-Met labeled proteins (Studts and Fox 1999) which lead to higher final biomass concentrations (OD600 of ~6 or ~11 for heavy isotope or Se-Met labeled proteins, respectively) and, thus higher labeled target protein yields.…”