Optimized Expression and Purification for High-Activity Preparations of Algal [FeFe]-Hydrogenase

Background: Recombinant expression and purification of metallo-enzymes, including hydrogenases, at high-yields is challenging due to complex, and enzyme specific, post-translational maturation processes. Low fidelities of maturation result in preparations containing a significant fraction of inactive, apo-protein that are not suitable for biophysical or crystallographic studies. Principal Findings: We describe the construction, overexpression and high-yield purification of a fusion protein consisting of the algal [2Fe2S]-ferredoxin PetF (Fd) and [FeFe]-hydrogenase HydA1. The maturation of Fd-HydA1 was optimized through improvements in culture conditions and media components used for expression. We also demonstrated that fusion of Fd to the N-terminus of HydA1, in comparison to the C-terminus, led to increased expression levels that were 4-fold higher. Together, these improvements led to enhanced HydA1 activity and improved yield after purification. The strong binding-affinity of Fd for DEAE allowed for two-step purification by ion exchange and StrepTactin affinity chromatography. In addition, the incorporation of a TEV protease site in the Fd-HydA1 linker allowed for the proteolytic removal of Fd after DEAE step, and purification of HydA1 alone by StrepTactin. In combination, this process resulted in HydA1 purification yields of 5 mg L−1 of culture from E. coli with specific activities of 1000 U (U = 1 µmol hydrogen evolved mg−1 min−1). Significance: The [FeFe]-hydrogenases are highly efficient enzymes and their catalytic sites provide model structures for synthetic efforts to develop robust hydrogen activation catalysts. In order to characterize their structure-function properties in greater detail, and to use hydrogenases for biotechnological applications, reliable methods for rapid, high-yield expression and purification are required.United States. Dept. of Energy. (contract DE-AC36-08-GO28308

Polypeptide Conjugates as High-affinity Binders for Proteins

A novel concept for protein recognition has been developed. The recognition unit is a hybrid molecule obtained by conjugation of a small organic molecule to a synthetic polypeptide selected from a 16-membered set of 42 amino acid residue sequences. The sequences are unordered and have no prior relation to the target proteins. The concept is based on the hypothesis that a small set of sequences capable of hydrophobic interactions, hydrogen bonding and electrostatic interactions can yield a binder for any selected protein, provided that the small molecule shows medium affinity or better and is reasonably selective. The concept has been illustrated by the design, synthesis and evaluation of binders for three different proteins, the C-reactive protein, CRP, human Carbonic anhydrase II, HCAII, and Acetylcholine esterase, AChE. Highly efficient binders for CRP have been developed by conjugation of a derivative of the natural ligand, phosphocholine, to the side chain of one of the amino acids in each polypeptide. The binders in the set show a wide range of affinities for CRP and the tightest binder, 4-C10L17-PC6, binds almost irreversibly. Selected binders have been evaluated in human serum, where they capture CRP with high selectivity.High-affinity binders have been developed for HCAII, and the selectivity evaluated by extraction of the protein from blood. The binder 4-C37L34-B, a polypeptide conjugated to a spacered benzenesulphonamide residue, was able to extract Carbonic anhydrases specifically and to discriminate between the two isoforms of human Carbonic anhydrase. The conjugation of an acridine derivative to a polypeptide via a 14 atom spacer has been shown to yield a binder with high affinity and selectivity for AChE. The selectivity was demonstrated by extraction of AChE from Cerebrospinal fluid. This thesis focuses on the development of a fast and reliable procedure for the construction, selection and evaluation of protein binders, with the ambition to develop a technology that is applicable to the development of binders for all proteins

Cell-free expression, purification, and ligand-binding analysis of Drosophila melanogaster olfactory receptors DmOR67a, DmOR85b and DmORCO

Insects transmit numerous devastating diseases, including malaria, dengue fever, and sleeping sickness. Olfactory cues guide insects to their hosts, and are thus responsible for disease transmission. Understanding the molecular basis of insect olfaction could facilitate the development of interventions. The first step is to heterologously overexpress and purify insect olfactory receptors (ORs). This is challenging, as ORs are membrane proteins. Here, we show that insect ORs and their co-receptor can be expressed in an E. coli cell-free system. After immunoaffinity chromatography, the ORs are similar to 95% pure, and up to 1 mg/10 ml reaction is obtained. Circular dichroism together with microscale thermophoresis indicate that each receptor is properly folded, and can bind its respective ligand. This is the first time insect ORs have been expressed in an E. coli system. The methods described here could facilitate future structure-function studies, which may aid in developments to alleviate the suffering of millions caused by insect-transmitted diseases.Funding Agencies|DARPA QuBE [N66001-10-1-4062]; Yang Trust Fund; WennerGren Foundation; Swedish Chemical Society; Claude Leon Foundation</p

The G protein coupled receptor CXCR4 designed by the QTY code becomes more hydrophilic and retains cell signaling activity

G protein-coupled receptors (GPCRs) are vital for diverse biological functions, including vision, smell, and aging. They are involved in a wide range of diseases, and are among the most important targets of medicinal drugs. Tools that facilitate GPCR studies or GPCR-based technologies or therapies are thus critical to develop. Here we report using our QTY (glutamine, threonine, tyrosine) code to systematically replace 29 membrane-facing leucine, isoleucine, valine, and phenylalanine residues in the transmembrane alpha-helices of the GPCR CXCR4. This variant, CXCR4(QTY29), became more hydrophilic, while retaining the ability to bind its ligand CXCL12. When transfected into HEK293 cells, it inserted into the cell membrane, and initiated cellular signaling. This QTY code has the potential to improve GPCR and membrane protein studies by making it possible to design functional hydrophilic receptors. This tool can be applied to diverse alpha-helical membrane proteins, and may aid in the development of other applications, including clinical therapies.Funding Agencies|Yang Trust Fund; WennerGren Foundation; Swedish Chemical Society; Claude Leon Foundation; University of the Witwatersrand; Wellcome Trust UKWellcome Trust</p

SDS-PAGE analysis of Fd-HydA1 expression, TEV digestion and HydA1 purification.

<p>(A). A comparison of the expression levels of N-terminal and C-terminal Fd-HydA1 fusions by separation on SDS-PAGE. Lanes show (left to right) protein size marker, SM, with sizes in kDa, and increasing amounts (0.5-to-4 µl) of total protein from cells expressing either the C-terminal (Fd C) or N-terminal (Fd N) Fd-HydA1 fusions. The location of ∼60 kDa Fd-HydA1 fusion, and 53 kDa HydG as an internal loading control are marked. The difference of Fd position on Fd-HydA1 expression levels was evident as a more intense brown color of the cell-lysates (inset image, top) harboring the N-terminal (right) versus C-terminal (left) Fd fusion. A Western Blot performed with antibodies to the StrepII-tag is shown (inset image, bottom) where the N-terminal fusion (right) exhibits a more intense band than the C-terminal fusion (left). (B). Analysis of TEV digestions of the N-terminal Fd-HydA1 fusion (30 µg) mixed with 1, 2, 4, or 8 µg of TEV (lanes 1–4, respectively). Lanes 2 and 3 show a partial digestion. The locations of Fd-HydA1 (60 kDa), HydA1 (50 kDa) and Fd (10 kDa) are indicated on the right. The optimal amount of TEV for complete digestion was 4 µg (shown in lane 3). Protein size-marker, SM, with sizes in kDa. (C). TEV digestions of DEAE pooled fractions containing Fd-HydA1 and separated on SDS-PAGE. The same w/w ratio of TEV to Fd-HydA1 was used as for purified Fd-HydA1 (4 µg TEV with 30 µg of DEAE pool). Lane 1, complete TEV digest; Lane 2, partial TEV digest; Lane 3, no TEV. Bands corresponding to Fd-HydA1 (60 kDa), HydA1 (50 kDa) and Fd (10 kDa) are identified on the right. HydG is identified as a loading control. (D). Analysis of HydA1 purity by SDS-PAGE. Protein size marker, SM, with sizes in kDa. Lanes 1–4 contain 1.4, 4, 6.8, and 12.3 µg, respectively, of purified HydA1 at >99% purity.</p

Buffers used for protein purification.

<p>Buffers used for protein purification.</p

Influence of growth factors on the <i>in vitro</i> hydrogen evolution activities in cells harboring pHydEFd-HydA1 and pHydFHydG.

<p>Hydrogenase activities (reported in units of nmol hydrogen evolved, ml⁻¹ of culture, min⁻¹) in solubilized whole-cells were measured as described in materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035886#s4" target="_blank">methods</a>. (A). The effect of the shaker rotation speed (RPM) during the aerobic growth phase was tested for 100 ml or 1 L culture volumes grown in. 250 ml or 2 L baffled flasks, respectively. The optimal rate was for both culture volumes was ∼300–350 RPM. (B). The effect of ferric ammonium citrate (FEC) concentration. Optimal levels were 2–2.5 mM. (C). Effect of IPTG concentration. Optimal levels of IPTG were 0.4 mM, lower than the 1.5 mM used previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035886#pone.0035886-Evans1" target="_blank">[13]</a>. (D). Effect of substituting Ampicillin with Carbenicillin during all stages of growth. Addition of Carbenicillin at 200 µg ml⁻¹ was required for peak hydrogenase activity levels.</p

HydA1 purification yields and specific activities from various expression hosts.

a<p>HydA1 specific activities for hydrogen evolution by MV assay; 1 U = µmol hydrogen min⁻¹ mg⁻¹.</p>b<p>HydA1 mg L⁻¹ of cell culture.</p>c<p>Iron content as mol iron (mol HydA1)⁻¹; NR, not reported.</p