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

Abstract

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.

Figure 1. The NESG Disorder Prediction Server (DisMeta) (http://www-nmr.cabm.rutgers.edu/bioinformatics/disorder/) and alternative constructs produced by the Construct Optimization Software for the Porphyromonas gingivalis protein Q7MX54 (NESG ID: PgR37)

(A) Output of the DisMeta server including prediction of secondary structure (PROFsec, PSIPred), parallel coiled coil regions (COIL) (Lupas et al., 1991), signal peptides (SignalP), transmembrane helices (TMHHM), low complexity regions (SEG) (Wootton and Federhen, 1996), and the Disorder Consensus plot showing the number of disorder prediction algorithms (0-8) predicting disorder versus the protein residue number in linear order N- to C- terminus. (B) Schematic representation of construct optimization for NESG protein target PgR37 from PFAM domain family DUF477, including: full-length, residues 54-187, residues 59-182 and residues 35-182. Only the 35-182 construct produced soluble expressed protein, and ultimately an NMR structure (Protein Data Bank ID:2KW7).

Figure 2. Schematic of the PLIMS directed high-throughput cloning process using the Qiagen BioRobot 8000

The PLIMS Database, utilizing a 96-well graphical user interface (GUI), generates DNA sequences corresponding to the protein regions selected by the NESG Construct Optimization Software. Primer Prim’er generates primer sets in 96 well format, each with the necessary 15 base pair overhangs for Infusion ligation-independent cloning. 96 well PCR reactions are performed, amplification products are visualized and separated by agarose gel electrophoresis. The results are archived in PLIMS and correct bands excised and purified using a custom 96 well-gel extraction protocol on the BioRobot. Purified amplification products contain 15-base pair regions of homology with each end of the linearized vector (shown as gray (5′) and black (3′) extensions on the PCR insert and vector in the lower center of the figure). Addition of the Infusion enzyme for pairing and resecting the region of overlap is followed by E. coli transformation. Colony PCR/agarose gel electrophoresis identifies the correct clones, the data is entered into PLIMS, which rearrays the colony PCR template plates for in inoculation of 48 well blocks and an automated 96 well miniprep protocol on the BioRobot.

Figure 3. High-throughput analytical scale protein expression screening using robotic methods

A 96-well transformation protocol is performed on the BioRobot. Colonies are transferred to an LB containing 2.2 mL 96-well S-Block (Qiagen), followed by overnight subculturing in MJ9 media (all manipulations performed on the BioRobot deck, shown in lower left). Plates are agitated in a microtiter plate freezing rack attached to a platform shaker (shown in the lower middle). A 1:20 dilution into four 24 well blocks (Qiagen) is performed and cells are grown to mid-log phase and induced overnight at 17 °C. Following overnight incubation cells are harvested by centrifugation (3000 g-force, 10 minutes) and resuspended in 100 μL of Lysis Buffer and transferred to a 96-well Round Bottom plate (Greiner). Following sonication (Qsonix 96 probe with cell lysate shown in upper right) a 30 μL aliquot of the total cellular lysate (Tot) is transferred to a new plate. The remainder is centrifuged for 10 minutes at 3000 g-force, and a 30 μL aliquot of the supernatant (Sol) is transferred to a new plate. SDS-PAGE analysis of equal amounts of Total cell extract (left) or Soluble cell extract (right): lane 1, SDS-PAGE standard (Precision Plus, Bio-Rad), lanes 2 and 3 NESG target HR6654C (residues 207- 289, 11 kDa), lanes 4 and 5 NESG target HR203 (residues 18-247, 26 kDa), lane 6 NESG target HR6430A (residues 287-370, 10.4 kDa). The constructs of all three human proteins produce soluble overexpressed protein of the correct size (highlighted with an asterisk).

Figure 4. 96-well midi-scale protein expression, purification, and characterization

This system utilizes (i) an Airlift Fermentation System (Genomics Institute of the Novartis Research Foundation - GNF) with O₂ aeration at 60 mL scale; (ii) a His MultiTrap HP 96-well plate (GE Healthcare) for Ni²⁺-affinity protein purification; and (iii) Zeba™ 96-well desalting spin plate (Thermo Scientific) for buffer exchange. Analytical protein chemistry and biophysical screening steps include target validation by MALDI-TOF mass spectrometry, concentration determination by a NanoDrop ND-8000 Spectrophotometer, homogeneity analysis by SDS-PAGE, Aggregation Screening by analytical gel filtration with static light scattering, and NMR screening (sample tubes loaded with Gilson 215 based automation) using a 1.7-mm micro cryo NMR probe and automated sample changer. Insets show representative results of ¹H NMR Screening (top right) and Aggregation Screening with static light scattering (lower right, M- monomer, D- dimer, O – oligomer, A – soluble aggregate, as explained in text).

Figure 5. Preparative-scale fermentation

(A) Flow chart of the preparative-scale fermentation process, The PLIMS database identifies targets suitable for fermentation and their expression glycerol stock location. The precultures and initial log-phase growth are carried out at 37 °C, overnight induction is performed at 17 °C. (B) SDS-PAGE analysis of a fermentation with four human protein targets gel showing Total Expressed (E - lanes 2, 4, 6, and 8) and Soluble (S – lanes 3, 5, 7, 9) extracts. The intensity of the targeted protein band in the total extract is used to estimate the total expression level (E), and the intensity of the band in the S extract is used to estimate the portion of the expressed protein that is soluble (S).

Figure 6. Construct optimization using amide hydrogen deuterium exchange with mass spectrometry detection (HDX-MS)

(A) Schematic of the HDX-MS process. Proteins are mixed with ²H₂O, exchange is quenched at three time points (e.g., 10 sec, 100 sec and 1000 sec) by lowering the pH and temperature. The samples are then subjected to a pepsin column for digestion, followed by reverse phase chromatography for peptide separation, and the degree of amide exchange prior to quenching is assessed by ESI-MS. (B) Results of the HDX-MS analysis for E. coli yiaD (NESG target ID: ER553). Primary sequence is shown at the top followed by PROFsec secondary structure prediction and heat map of the HDX-MS results at 10, 100 and 1000 sec reaction intervals. Note that the numbering does not include the N-terminal lipoprotein signal sequence that is cleaved from the mature protein. (C) ¹H-¹⁵N HSQC spectra of full length (left) and construct optimized (right) E. coli yiaD (59-199).