Stephen Anderson

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

SummaryAn 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 13C6-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 13C6-glucose usage. Incorporation levels of 13C and/or 15N isotopes in purified 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.

The high-throughput protein sample production platform of the Northeast Structural Genomics Consortium

We describe the core Protein Production Platform of the Northeast Structural Genomics Consortium (NESG) and outline the strategies used for producing high-quality protein samples. The platform is centered on the cloning, expression and purification of 6X-His-tagged proteins using T7-based Escherichia coli systems. The 6X-His tag allows for similar purification procedures for most targets and implementation of high-throughput (HTP) parallel methods. In most cases, the 6X-His-tagged proteins are sufficiently purified (>97% homogeneity) using a HTP two-step purification protocol for most structural studies. Using this platform, the open reading frames of over 16,000 different targeted proteins (or domains) have been cloned as>26,000 constructs. Over the past 10 years, more than 16,000 of these expressed protein, and more than 4400 proteins (or domains) have been purified to homogeneity in tens of milligram quantities (see Summary Statistics, http://nesg.org/statistics.html). Using these samples, the NESG has deposited more than 900 new protein structures to the Protein Data Bank (PDB). The methods described here are effective in producing eukaryotic and prokaryotic protein samples in E. coli. This paper summarizes some of the updates made to the protein production pipeline in the last 5 years, corresponding to phase 2 of the NIGMS Protein Structure Initiative (PSI-2) project. The NESG Protein Production Platform is suitable for implementation in a large individual laboratory or by a small group of collaborating investigators. These advanced automated and/or parallel cloning, expression, purification, and biophysical screening technologies are of broad value to the structural biology, functional proteomics, and structural genomics communities.

Preparation of protein samples for NMR structure, function, and small-molecule screening studies.

In this chapter, we concentrate on the production of high-quality protein samples for nuclear magnetic resonance (NMR) studies. In particular, we provide an in-depth description of recent advances in the production of NMR samples and their synergistic use with recent advancements in NMR hardware. We describe the protein production platform of the Northeast Structural Genomics Consortium and outline our high-throughput strategies for producing high-quality protein samples for NMR studies. Our strategy is based on the cloning, expression, and purification of 6×-His-tagged proteins using T7-based Escherichia coli systems and isotope enrichment in minimal media. We describe 96-well ligation-independent cloning and analytical expression systems, parallel preparative scale fermentation, and high-throughput purification protocols. The 6×-His affinity tag allows for a similar two-step purification procedure implemented in a parallel high-throughput fashion that routinely results in purity levels sufficient for NMR studies (>97% homogeneity). Using this platform, the protein open reading frames of over 17,500 different targeted proteins (or domains) have been cloned as over 28,000 constructs. Nearly 5000 of these proteins have been purified to homogeneity in tens of milligram quantities (see Summary Statistics, http://nesg.org/statistics.html), resulting in more than 950 new protein structures, including more than 400 NMR structures, deposited in the Protein Data Bank. The Northeast Structural Genomics Consortium pipeline has been effective in producing protein samples of both prokaryotic and eukaryotic origin. Although this chapter describes our entire pipeline for producing isotope-enriched protein samples, it focuses on the major updates introduced during the last 5 years (Phase 2 of the National Institute of General Medical Sciences Protein Structure Initiative). Our advanced automated and/or parallel cloning, expression, purification, and biophysical screening technologies are suitable for implementation in a large individual laboratory or by a small group of collaborating investigators for structural biology, functional proteomics, ligand screening, and structural genomics research.

A High Through-put Platform for Recombinant Antibodies to Folded Proteins

Antibodies are key reagents in biology and medicine, but commercial sources are rarely recombinant and thus do not provide a permanent and renewable resource. Here, we describe an industrialized platform to generate antigens and validated recombinant antibodies for 346 transcription factors (TFs) and 211 epigenetic antigens. We describe an optimized automated phage display and antigen expression pipeline that in aggregate produced about 3000 sequenced Fragment antigen-binding domain that had high affinity (typically EC50<20 nm), high stability (Tm∼80 °C), good expression in E. coli (∼5 mg/L), and ability to bind antigen in complex cell lysates. We evaluated a subset of Fabs generated to homologous SCAN domains for binding specificities. These Fragment antigen-binding domains were monospecific to their target SCAN antigen except in rare cases where they cross-reacted with a few highly related antigens. Remarkably, immunofluorescence experiments in six cell lines for 270 of the TF antigens, each having multiple antibodies, show that ∼70% stain predominantly in the cytosol and ∼20% stain in the nucleus which reinforces the dominant role that translocation plays in TF biology. These cloned antibody reagents are being made available to the academic community through our web site recombinant-antibodies.org to allow a more system-wide analysis of TF and chromatin biology. We believe these platforms, infrastructure, and automated approaches will facilitate the next generation of renewable antibody reagents to the human proteome in the coming decade.

Nucleosome interaction surface of linker histone H1c is distinct from that of H1(0).

The fully organized structure of the eukaryotic nucleosome remains unsolved, in part due to limited information regarding the binding site of the H1 or linker histone. The central globular domain of H1 is believed to interact with the nucleosome core at or near the dyad and to bind at least two strands of DNA. We utilized site-directed mutagenesis and in vivo photobleaching to identify residues that contribute to the binding of the globular domain of the somatic H1 subtype H1c to the nucleosome. As was previously observed for the H10 subtype, the binding residues for H1c are clustered on the surface of one face of the domain. Despite considerable structural conservation between the globular domains of these two subtypes, the locations of the binding sites identified for H1c are distinct from those of H10. We suggest that the globular domains of these two linker histone subtypes will bind to the nucleosome with distinct orientations that may contribute to higher order chromatin structure heterogeneity or to differences in dynamic interactions with other DNA or chromatin-binding proteins.

Structural alteration of cofactor specificity in Corynebacterium 2,5-diketo-D-gluconic acid reductase

Corynebacterium 2,5‐Diketo‐D‐gluconic acid reductase (2,5‐DKGR) catalyzes the reduction of 2,5‐diketo‐D‐gluconic acid (2,5‐DKG) to 2‐Keto‐L‐gulonic acid (2‐KLG). 2‐KLG is an immediate precursor to L‐ascorbic acid (vitamin C), and 2,5‐DKGR is, therefore, an important enzyme in a novel industrial method for the production of vitamin C. 2,5‐DKGR, as with most other members of the aldo‐keto reductase (AKR) superfamily, exhibits a preference for NADPH compared to NADH as a cofactor in the stereo‐specific reduction of substrate. The application of 2,5‐DKGR in the industrial production of vitamin C would be greatly enhanced if NADH could be efficiently utilized as a cofactor. A mutant form of 2,5‐DKGR has previously been identified that exhibits two orders of magnitude higher activity with NADH in comparison to the wild‐type enzyme, while retaining a high level of activity with NADPH. We report here an X‐ray crystal structure of the holo form of this mutant in complex with NADH cofactor, as well as thermodynamic stability data. By comparing the results to our previously reported X‐ray structure of the holo form of wild‐type 2,5‐DKGR in complex with NADPH, the structural basis of the differential NAD(P)H selectivity of wild‐type and mutant 2,5‐DKGR enzymes has been identified.

Does the Kunitz domain from the Alzheimer's amyloid β protein precursor inhibit a kallikrein responsible for post-translational processing of nerve growth factor precursor?

Alternative splicing of the Alzheimers amyloid β protein precursor (ABPP) message leads to the production of several variants of this precursor polypeptide. Two of these variants contain a domain that is highly homologous to members of the Kunitz class of protease inhibitors. In order to initiate a study of the physiological role of this domain, we have produced active ABPP Kunitz inhibitor by constructing and expressing a synthetic gene in E. coli. Nerve growth factor (NGF) deficiency has been suggested as a possible cause of the neural degeneration characteristic of Alzheimers disease, and trypsin and γ‐NGF are the two enzymes that have been shown to be capable of processing β‐NGF precursor to active, mature β‐NGF in vitro, therefore the specificity of purified recombinant ABPP Kunitz inhibitor was analyzed with respect to these two proteases. Binding of isolated ABPP Kunitz domain both to trypsin (K i,app < 10 nM and to γ‐NGF (K i,app = 300 nM) was observed. This difference in binding to the two proteases correlates with the approximately 20‐fold higher rate observed for in vitro processing of the β‐NGF precursor by trypsin compared to processing by γ‐NGF, indicating that perhaps the inhibitor mimics the interaction of the β‐NGF precursor with proteases. The kallikrein actually responsible for β‐NGF precursor processing in vivo is unknown, but these results suggest that it is capable of being significantly inhibited by exposure to the ABPP Kunitz domain.

Molecular Modeling of Substrate Binding in Wild-Type and Mutant Corynebacteria 2,5-Diketo-D-gluconate Reductases

2,5‐Diketo‐D‐gluconic acid reductase (2,5‐DKGR; E.C. 1.1.1.‐) catalyzes the Nicotinamide adenine dinucleotide phosphate (NADPH)‐dependent stereo‐specific reduction of 2,5‐diketo‐D‐gluconate (2,5‐DKG) to 2‐keto‐L‐gulonate (2‐KLG), a precursor in the industrial production of vitamin C (L‐ascorbate). Microorganisms that naturally ferment D‐glucose to 2,5‐DKG can be genetically modified to express the gene for 2,5‐DKGR, and thus directly produce vitamin C from D‐glucose. Two naturally occurring variants of DKGR (DKGR A and DKGR B) have been reported. DKGR B exhibits higher specific activity toward 2,5‐DKG than DKGR A; however, DKGR A exhibits a greater selectivity for this substrate and significantly higher thermal stability. Thus, a modified form of DKGR, combining desirable properties from both enzymes, would be of substantial commercial interest. In the present study we use a molecular dynamics–based approach to understand the conformational changes in DKGR A as the active site is mutated to include two active site residue changes that occur in the B form. The results indicate that the enhanced kinetic properties of the B form are due, in part, to residue substitutions in the binding pocket. These substitutions augment interactions with the substrate or alter the alignment with respect to the putative proton donor group. Proteins 2000;39:68–75.

Verification of a Novel NADH-Binding Motif: Combinatorial Mutagenesis of Three Amino Acids in the Cofactor-Binding Pocket of Corynebacterium 2,5-Diketo-D-Gluconic Acid Reductase

A screening method has been developed to support randomized mutagenesis of amino acids in the cofactor-binding pocket of the NADPH-dependent 2,5-diketo-D-gluconic acid (2,5-DKG) reductase. Such an approach could enable the isolation of an enzyme that can better catalyze the reduction of 2,5-DKG to 2-keto-L-gulonic acid (2-KLG) using NADH as a cofactor. 2-KLG is a valuable precursor to ascorbic acid, or vitamin C, and an enzyme with increased activity with NADH may be able to improve two potential vitamin C production processes. Previously we have identified three amino acid residues that can be mutated to improve activity with NADH as a cofactor. As a pilot study to show feasibility, a library was made with these three amino acids randomized, and 300 random colonies were screened for increased NADH activity. The activities of seven mutants with apparent improvements were verified using activity-stained native gels, and sequencing showed that the amino acids obtained were similar to some of those already discovered using rational design. The four most active mutants were purified and kinetically characterized. All of the new mutations resulted in apparent kcat values that were equal to or higher than that of the best mutant obtained through rational design. At saturating levels of cofactor, the best mutant obtained was almost twice as active with NADH as a cofactor as the wild-type enzyme is with NADPH. This screen is a valuable tool for improving 2,5-DKG reductase, and it could easily be modified for improving other aspects of this protein or similar enzymes.

A Biocatalytic Approach to Vitamin C Production

Although the primary focus of the biotechnology industry has been on the overproduction of new proteins, primarily for pharmaceutical purposes, there has been growing interest in other fields such as agriculture, diagnostics, and the biocatalytic production of organic chemicals. We have recently been working to develop a novel biosynthetic process for the production of vitamin C (L-ascorbic acid, ASA). This approach has involved the study of the enzymes, coenzymes, and metabolic pathways of different bacteria with the goal of creating new metabolic routes to make a new product (Fig. 6.1). This metabolic pathway engineering approach, which required the identification and characterization of several new enzymes and the cloning and expression of the gene coding for one of these enzymes, has led to a successful one-step bioconversion of D-glucose (G) into 2-keto-L-gulonic acid (2-KLG), a key intermediate in the synthesis of ascorbic acid.