Rapid, robotic, small-scale protein production for NMR screening and structure determination

Abstract

Three-dimensional protein structure determination is a costly process due in part to the low success rate within groups of potential targets. Conventional validation methods eliminate the vast majority of proteins from further consideration through a time-consuming succession of screens for expression, solubility, purification, and folding. False negatives at each stage incur unwarranted reductions in the overall success rate. We developed a semi-automated protocol for isotopically-labeled protein production using the Maxwell-16, a commercially available bench top robot, that allows for single-step target screening by 2D NMR. In the span of a week, one person can express, purify, and screen 48 different (15)N-labeled proteins, accelerating the validation process by more than 10-fold. The yield from a single channel of the Maxwell-16 is sufficient for acquisition of a high-quality 2D (1)H-(15)N-HSQC spectrum using a 3-mm sample cell and 5-mm cryogenic NMR probe. Maxwell-16 screening of a control group of proteins reproduced previous validation results from conventional small-scale expression screening and large-scale production approaches currently employed by our structural genomics pipeline. Analysis of 18 new protein constructs identified two potential structure targets that included the second PDZ domain of human Par-3. To further demonstrate the broad utility of this production strategy, we solved the PDZ2 NMR structure using [U-(15)N,(13)C] protein prepared using the Maxwell-16. This novel semi-automated protein production protocol reduces the time and cost associated with NMR structure determination by eliminating unnecessary screening and scale-up steps.

Figure 1

Optimization of the Maxwell-16 buffer conditions and workflow. (a) IMAC purification is performed in a seven-well cartridge. Magnetic IMAC beads are conveyed by a magnetic plunger that also serves as a mixing device. Cell lysis is accomplished by the addition of FastBreak cell lysis buffer and gentle agitation by the plunger in well 1. The plunger retrieves the MagneHis resin from well 2 and returns to well one to adsorb the His-tagged protein. The bound protein undergoes four washes in wells 3–6. The purified protein is deposited in a single cuvette at the end of the procedure. The binding, wash and elution steps are repeated once in every Maxwell-16 purification. (b) Flowchart comparing conventional and automated protein purification workflows. In a conventional two-stage scheme for target screening, a workgroup of 24 targets is transformed, cultured, and evaluated for soluble expression by SDS-PAGE during week 1. In week 2, up to 8 targets may be selected for large-scale expression in ¹⁵N-enriched minimal medium, purification and 2D ¹H-¹⁵N HSQC analysis. The optimized Maxwell-16 protocol parallelizes the protein purification process using 60 mL cultures, enabling a single technician to process up to 48 targets/week, reducing the total time for NMR screening by half, and increasing overall throughput by ∼10-fold. (c) Protein yields were enhanced by optimizing the volume of beads, lysis buffer, and elution buffer used. Additionally, we investigated how the elution buffer pH and imidazole concentration influenced release of protein from the MagneHis Ni-resin. SDS-PAGE analysis of the three test proteins revealed target-to-target variations. However, increasing the volume of MagneHis Ni-resin to 150 μL and lowering the pH of the elution buffer to 6.5 yielded the most consistent results. All studies were conducted with culture volumes corresponding to a total OD₆₀₀ = 60.

Figure 2

Two-dimensional NMR analysis of control workgroup proteins. Samples from the control workgroup were subject to ¹H-¹⁵N HSQC NMR. Spectra were acquired in ∼80 min using 16 transients per FID at 25°C on a Bruker Avance 600 MHz NMR equipped with a 5 mm TCI Cryoprobe. Purity of samples generated by the Maxwell-16 is illustrated by SDS-PAGE (inset). Each gel contains samples of depleted lysate (DL), each wash step (W1–W4) and pure protein in the elution cuvette (EC) prior to exchange into the selected NMR buffer.

Figure 3

Two-dimensional NMR analysis of hPar-3 and β-PIX domain constructs. All 18 of the hPar-3 and β-PIX constructs were analyzed by ¹H-¹⁵N HSQC NMR. Spectra were acquired in ∼80 min using 16 transients per FID at 25°C on a Bruker Avance 600 MHz NMR equipped with a 5 mm TCI Cryoprobe. HSQC spectra for constructs that are folded, partially folded or unfolded are labeled in black and those that contain no signal are labeled in gray. The two proteins that are suitable for NMR structure determination are indicated with an asterisk.

Figure 4

Validation of the optimized Maxwell-16 protocol. (a) An assigned ¹H-¹⁵N-HSQC of hPar3-PDZ2 shows 113 of 114 expected resonances. Data collection was done at 25°C in 3 mm sample cells with 4 transients per FID using a 1 mM sample of hPar3 PDZ2 in 20 mM sodium phosphate pH 6.5 containing 50 mM sodium chloride, 0.02% sodium azide, and 10% (v/v) ²H₂O. (b) Comparison of four ¹⁵N-edited 3D NOESY-HSQC strips from spectra collected on hPar3 PDZ2 (1 mM) prepared by a conventional large-scale production protocol (left) or the Maxwell-16 protocol (right). Data for the standard sample was acquired in a 5-mm Shigemi tube at 600 MHz, whereas data collection for the Maxwell-16 sample was acquired in a 3-mm NMR tube at 500 MHz with equal data collection times. Spectra of equivalent or higher quality were obtained from the Maxwell-16 sample despite a 2.5-fold reduction in the active NMR sample by mass and volume. Similar results were observed for the 3D triple-resonance experiments. (c) Ribbon diagram of the hPar3 PDZ2 NMR structure solved protein generated by five channels of the Maxwell-16. Structural statistics for the NMR ensemble are presented in Table III.