Experimental mapping of soluble protein domains using a hierarchical approach

Abstract

Exploring the function and 3D space of large multidomain protein targets often requires sophisticated experimentation to obtain the targets in a form suitable for structure determination. Screening methods capable of selecting well-expressed, soluble fragments from DNA libraries exist, but require the use of automation to maximize chances of picking a few good candidates. Here, we describe the use of an insertion dihydrofolate reductase (DHFR) vector to select in-frame fragments and a split-GFP assay technology to filter-out constructs that express insoluble protein fragments. With the incorporation of an IPCR step to create high density, focused sublibraries of fragments, this cost-effective method can be performed manually with no a priori knowledge of domain boundaries while permitting single amino acid resolution boundary mapping. We used it on the well-characterized p85α subunit of the phosphoinositide-3-kinase to demonstrate the robustness and efficiency of our methodology. We then successfully tested it onto the polyketide synthase PpsC from Mycobacterium tuberculosis, a potential drug target involved in the biosynthesis of complex lipids in the cell envelope. X-ray quality crystals from the acyl-transferase (AT), dehydratase (DH) and enoyl-reductase (ER) domains have been obtained.

Figure 1.

The GFP-enabled domain trapping strategy. The PCR-amplified gene is fragmented by chemical or mechanical means and DNA fragments of desired size are excised from agarose gel. Blunt-end fragments are cloned into the iDHFR ORF filter, where only the in-frame ones permitting the expression of the second half of DHFR will survive. Inserts from the recovered plasmids are cloned into the split-GFP vector and used for IPCR to create high density, focused sublibraries of fragments prior to the split-GFP assay. A range of fluorescent clones are picked and grown in 96-well liquid cultures for in vitro quantification of the soluble and insoluble protein fractions. Clones are sequenced and the fragments are aligned onto the full parent gene. Fragments can be directly tested for expression or subcloned without the S11 tag into a pET vector. Numbers to the left indicate the approximate library size at the different steps.

Figure 2.

Screening clones using the split-GFP reassembly assay. Images showing cell colony fluorescence from agar plates after sequential induction (solubility reporter) and co-induction (expression reporter) of the GFP 11-tagged protein fragments and its complementary GFP 1–10 detector. Clones displaying a wide range of fluorescence are visible after sequential induction.

Figure 3.

Mapping of the IPCR p85α targeted fragments. IPCRs using oppositely directed primers were used to generate sublibraries of fragments. For each sublibrary, an ensemble of 96 clones with a wide range of in vivo fluorescence intensities were picked and grown in 96-well liquid culture plates for in vitro split-GFP solubility screen. Only the correctly sequenced in-frame fragments are represented. Based on the in vitro solubility assays, fragments were color-coded black, light green and bright green, where the black side of the spectrum identifies the bottom 20% least soluble protein fragments and the bright green side corresponds to the top 20% most soluble ones. IPCRs within p85α were used to generate four large-size sublibraries (350–750 kb) centered onto the SH3, BCR, N–SH2 and C–SH2 domains. Within each library, solubility values from three or more identical fragments were averaged in order to keep the color-coded representation as clear as possible. Boundaries of structure solved domains are indicated in red and the junction at amino acid position 9 is indicated in blue.

Figure 4.

PCR-directed truncations of the p85α BCR domain. (a) Schematic representation of all 144 constructs aligned onto the p85α amino-acid sequence. N- and C-terminal positions are indicated. Fragments are organized in groups of 12 with identical N-terminal positions. (b) Expression (left) and solubility (right) levels of E. coli BL21(DE3) cells expressing the corresponding fragments in fusion with S11 following complementation with GFP 1–10. Orange dashed lines mark boundaries, where dramatic changes in expression and solubility levels were observed. (c) In addition to 105–319 for which the X-ray structure is known (PDB code: 1PBW), well-expressed and soluble fragments were selected for downstream NMR studies. HSQC spectra of fragments 105–299 (blue), 105–309 (red), 105–319 (black with corresponding white rectangle in the solubility screen) and 115–299 (green) are represented.

Figure 5.

Mapping of the IPCR PpsC targeted fragments. Five large-size sublibraries of fragments (850–1650 kb) centered onto the KS, AT, DH, ER and ACP domains were generated. Picking and color coding of fragments follow the same rules as in Figure 3. Red dots indicate the fragments selected for downstream biochemical and structural characterization.

Figure 6.

Expression, solubility and crystallization trials of selected PpsC fragments. (a) Ten fragments covering the structural domains of PpsC were subcloned from the pTET-GFP 11 plasmid into a N6–HIS pET vector for biochemical and biophysical characterization. (b) SDS–PAGE of soluble (S) and pellet (P) fractions of E. coli BL21 (DE3) cells expressing the different fragments. (c) Pictures of X-ray quality crystals of selected fragments represented as a black rectangle in (a) are shown. The 3D structures of the AT (545–877), DH (921–1222), and truncated ER (1558–1750) domains have been recently determined at 1.8, 2.8, and 2.5 Å resolutions, respectively.