Effects of human versus mouse leukemia inhibitory factor on the in vitro development of bovine embryos

Leukemia inhibitory factor (LIF) is a cytokine that shows conflicting effects on in vitro produced (IVP) bovine embryos. Bovine LIF (bLIF) has been cloned and used in culture, but there is no commercially available bLIF. Thus, researchers use human LIF (hLIF) to supplement the culture medium for bovine embryos because of its greater sequence homology compared to murine LIF (mLIF). We compared the effects of mLIF and hLIF on the development of bovine embryos in culture with the effects described for bLIF. Oocytes were matured and fertilized in vitro and cultured in modified synthetic oviduct fluid with BSA. On Day 6 post-insemination, morulae were cultured for 48h in the presence of: (1) mLIF, 100ngml(-1); (2) hLIF, 100ngml(-1); or (3) no LIF. Reduced blastocyst rates were observed on Day 8 for hLIF at the middle and expanded stages, while mLIF had no effect. In contrast, Day 8 blastocysts showed decreased cell counts both in terms of inner cell mass (ICM) and ICM/total cell proportions in the presence of mLIF, while hLIF had no effect. No changes were seen in trophectoderm (TE) and total cell counts. The increased hatching rates and TE cell counts previously described for bLIF, together with the disparate effects exhibited by hLIF and mLIF during blastocyst formation indicate these compounds are inappropriate to replace bLIF. We recommend that heterospecific LIF should not be used to supplement the culture medium for bovine embryo or embryonic stem cells.     

Structure-function analysis of human IL-6: identification of two distinct regions that are important for receptor binding.

Interleukin-6 (IL-6) is a multifunctional cytokine that plays an important role in host defense. It has been predicted that IL-6 may fold as a 4 alpha-helix bundle structure with up-up-down-down topology. Despite a high degree of sequence similarity (42%) the human and mouse IL-6 polypeptides display distinct species-specific activities. Although human IL-6 (hIL-6) is active in both human and mouse cell assays, mouse IL-6 (mIL-6) is not active on human cells. Previously, we demonstrated that the 5 C-terminal residues of mIL-6 are important for activity, conformation, and stability (Ward LD et al., 1993, Protein Sci 2:1472-1481). To further probe the structure-function relationship of this cytokine, we have constructed several human/mouse IL-6 hybrid molecules. Restriction endonuclease sites were introduced and used to ligate the human and mouse sequences at junction points situated at Leu-62 (Lys-65 in mIL-6) in the putative connecting loop AB between helices A and B, at Arg-113 (Val-117 in mIL-6) at the N-terminal end of helix C, at Lys-150 (Asp-152 in mIL-6) in the connecting loop CD between helices C and D, and at Leu-178 (Thr-180 in mIL-6) in helix D. Hybrid molecules consisting of various combinations of these fragments were constructed, expressed, and purified to homogeneity. The conformational integrity of the IL-6 hybrids was assessed by far-UV CD. Analysis of their biological activity in a human bioassay (using the HepG2 cell line), a mouse bioassay (using the 7TD1 cell line), and receptor binding properties indicates that at least 2 regions of hIL-6, residues 178-184 in helix D and residues 63-113 in the region incorporating part of the putative connecting loop AB through to the beginning of helix C, are critical for efficient binding to the human IL-6 receptor. For human IL-6, it would appear that interactions between residues Ala-180, Leu-181, and Met-184 and residues in the N-terminal region may be critical for maintaining the structure of the molecule; replacement of these residues with the corresponding 3 residues in mouse IL-6 correlated with a significant loss of alpha-helical content and a 200-fold reduction in activity in the mouse bioassay. A homology model of mIL-6 based on the X-ray structure of human granulocyte colony-stimulating factor is presented.     

Affinity maturation of leukemia inhibitory factor and conversion to potent antagonists of signaling

Leukemia inhibitory factor (LIF)-induced cell signaling occurs following sequential binding to the LIF receptor alpha-chain (LIFR), then to the gp130 co-receptor used by all members of the interleukin-6 family of cytokines. By monovalently displaying human LIF on the surface of M13 phage and randomizing clusters of residues in regions predicted to be important for human LIFR binding, we have identified mutations, which lead to significant increases in affinity for binding to LIFR. Six libraries were constructed in which regions of 4-6 amino acids were randomized then panned against LIFR. Mutations identified in three distinct clusters, residues 53-57, 102-103, and 150-155, gave rise to proteins with significantly increased affinity for binding to both human and mouse LIFR. Combining the mutations for each of these regions further increased the affinity, such that the best mutants bound to human LIFR with >1000-fold higher affinity than wild-type human LIF. NMR analysis indicated that the mutations did not alter the overall structure of the molecule relative to the native protein, although some local changes occurred in the vicinity of the substituted residues. Despite increases in LIFR binding affinity, these mutants did not show any increase in activity as agonists of LIF-induced proliferation of Ba/F3 cells expressing human LIFR and gp130 compared with wild-type LIF. Incorporation of two additional mutations (Q29A and G124R), which were found to abrogate cell signaling, led to the generation of highly potent antagonists of both human and murine LIF-induced bioactivity.     

Murine recombinant leukemia inhibitory factor modulates inhibitory effect of 1,25 dihydroxyvitamin D3 on alkaline phosphatase activity in MC3T3-E1 cells

We demonstrated murine leukemia inhibitory factor (mLIF) mRNA in osteoblastic MC3T3-E1 cells, but not mLIF in their conditioned medium. Recombinant mLIF had an inhibitory effect on alkaline phosphatase (ALP) activity, but not on DNA synthesis, in these mLIF-free cells. This inhibitory effect was not prostaglandin E2 dependent. mLIF also modulated the inhibitory effect of 1,25 dihydroxyvitamin D3 [1,25(OH)2D3] on ALP activity, partly via down regulation of 1,25(OH)2D3 binding sites. These results suggest that LIF may play a role in regulating osteoblast differentiation.     

Mutations in the immunoglobulin-like domain of gp190, the leukemia inhibitory factor (LIF) receptor,increase or decrease its affinity for LIF

The leukemia inhibitory factor (LIF) receptor comprises the low affinity binding chain gp190 and the high affinity converter gp130. The ectodomain of gp190 is among the most complex in the hematopoietin receptor family, because it contains two typical cytokine receptor homology domains separated by an immunoglobulin-like (Ig-like) domain. Human and murine gp190 proteins share 76% homology, but murine gp190 binds human LIF with a much higher affinity, a property attributed to the Ig-like domain. Using alanine-scanning mutagenesis of the Ig-like domain, we mapped a LIF binding site at its carboxyl terminus, mainly involving residue Phe-328. Mutation of selected residues into their orthologs in the murine receptor (Q251E and N321D) significantly increased the affinity for human LIF. Interestingly, these residues, although localized at both the amino and carboxyl terminus, make a spatially unique LIF binding site in a structural model of the Ig-like module. These results demonstrate definitively the role of the Ig-like domain in LIF binding and the potential to modulate receptor affinity in this family with very limited amino acid changes.     

Systematic mutational mapping of sites on human interferon-beta-1a that are important for receptor binding and functional activity

A systematic mutational analysis of human interferon-beta-1a (IFN-beta) was performed to identify regions on the surface of the molecule that are important for receptor binding and for functional activity. The crystal structure of IFN-beta-1a was used to design a panel of 15 mutant proteins, in each of which a contiguous group of 2-8 surface residues was mutated, in most instances to alanine. The mutants were analyzed for activity in vitro in antiviral and in antiproliferation assays, and for their ability to bind to the type I IFN (ifnar1/ifnar2) receptor on Daudi cells and to a soluble ifnar2 fusion protein (ifnar2-Fc). Abolition of binding to ifnar2-Fc for mutants A2, AB1, AB2, and E established that the ifnar2 binding site on IFN-beta comprises parts of the A helix, the AB loop, and the E helix. Mutations in these areas, which together define a contiguous patch of the IFN-beta surface, also resulted in reduced affinity for binding to the receptor on cells and in reductions in activity of 5-50-fold in functional assays. A second receptor interaction site, concluded to be the ifnar1 binding site, was identified on the opposite face of the molecule. Mutations in this region, which encompasses parts of the B, C, and D helices and the DE loop, resulted in disparate effects on receptor binding and on functional activity. Analysis of antiproliferation activity as a function of the level of receptor occupancy allowed mutational effects on receptor activation to be distinguished from effects on receptor binding. The results suggest that the binding energy from interaction of IFN-beta with ifnar2 serves mainly to stabilize the bound IFN/receptor complex, whereas the binding energy generated by interaction of certain regions of IFN-beta with ifnar1 is not fully expressed in the observed affinity of binding but instead serves to selectively stabilize activated states of the receptor.     

Identification of residues important both for primary receptor binding and specificity in fibroblast growth factor-7

Fibroblast growth factors (FGFs) mediate a multitude of physiological and pathological processes by activating a family of tyrosine kinase receptors (FGFRs). Each FGFR binds to a unique subset of FGFs and ligand binding specificity is essential in regulating FGF activity. FGF-7 recognizes one FGFR isoform known as the FGFR2 IIIb isoform or keratinocyte growth factor receptor (KGFR), whereas FGF-2 binds well to FGFR1, FGFR2, and FGFR4 but interacts poorly with KGFR. Previously, mutations in FGF-2 identified a set of residues that are important for high affinity receptor binding, known as the primary receptor-binding site. FGF-7 contains this primary site as well as a region that restricts interaction with FGFR1. The sequences that confer on FGF-7 its specific binding to KGFR have not been identified. By utilizing domain swapping and site-directed mutagenesis we have found that the loop connecting the beta4-beta5 strands of FGF-7 contributes to high affinity receptor binding and is critical for KGFR recognition. Replacement of this loop with the homologous loop from FGF-2 dramatically reduced both the affinity of FGF-7 for KGFR and its biological potency but did not result in the ability to bind FGFR1. Point mutations in residues comprising this loop of FGF-7 reduced both binding affinity and biological potency. The reciprocal loop replacement mutant (FGF2-L4/7) retained FGF-2 like affinity for FGFR1 and for KGFR. Our results show that topologically similar regions in these two FGFs have different roles in regulating receptor binding specificity and suggest that specificity may require the concerted action of distinct regions of an FGF.     

Defining the structural basis for assembly of a transmembrane cytochrome

To define the structural basis for cofactor binding to membrane proteins, we introduce a manageable model system, which allows us, for the first time, to study the influence of individual transmembrane helices and of single amino acid residues on the assembly of a transmembrane cytochrome. In vivo as well as in vitro analyses indicate central roles of single amino acid residues for either interaction of the transmembrane helices or for binding of the cofactor. The results clearly show that interaction of the PsbF transmembrane helix is independent from binding of the heme cofactor. On the other hand, binding of the cofactor highly depends on helix-helix interactions. By site-directed mutagenesis critical amino acid residues were identified, which are involved in the assembly of a functional transmembrane cytochrome. Especially, a highly conserved glycine residue is critical for interaction of the transmembrane helices and assembly of the cytochrome. Based on the two-stage-model of alpha-helical membrane protein folding, the presented results clearly indicate a third stage of membrane protein folding, in which a cofactor binds to a pre-assembled transmembrane protein.     

Biochemical characterization of the high affinity binding between the glycine receptor and gephyrin

Gephyrin is an essential and instructive molecule for the formation of inhibitory synapses. Gephyrin binds directly to the large cytoplasmic loop located between transmembrane helices three and four of the beta-subunit of the glycine receptor and to microtubules, thus promoting glycine receptor (GlyR) anchoring to the cytoskeleton and clustering in the postsynaptic membrane. Besides its structural role, gephyrin is involved in the biosynthesis of the molybdenum cofactor that is essential for all molybdenum-dependent enzymes in mammals. Gephyrin can be divided into an N-terminal trimeric G domain and a C-terminal E domain, which are connected by a central linker region. Here we have studied the in vitro interaction of gephyrin and its domains with the large cytoplasmic loop of the GlyR beta-sub-unit (GlyRbeta-loop). Binding of gephyrin to the GlyR is exclusively mediated by the E domain, and the binding site was mapped to one of its sub-domains (residues 496-654). By using isothermal titration calorimetry, a high affinity (K(d) = 0.2-0.4 microm) and low affinity (K(d) = 11-30 microm) binding site for the GlyRbeta-loop was found on holo-gephyrin and the E domain, respectively, with a binding stoichiometry of two GlyRbeta-loops per E domain in both cases. Binding of the GlyRbeta-loop does not change the oligomeric state of either full-length gephyrin or the isolated E domain.     

Identification of residues at the alpha and epsilon subunit interfaces mediating species selectivity of Waglerin-1 for nicotinic acetylcholine receptors.

Waglerin-1 (Wtx-1) is a 22-amino acid peptide that is a competitive antagonist of the muscle nicotinic receptor (nAChR). We find that Wtx-1 binds 2100-fold more tightly to the alpha-epsilon than to the alpha-delta binding site interface of the mouse nAChR. Moreover, Wtx-1 binds 100-fold more tightly to the alpha-epsilon interface from mouse nAChR than that from rat or human sources. Site-directed mutagenesis of residues differing in the extracellular domains of rat and mouse epsilon subunits indicates that residues 59 and 115 mediate the species difference in Wtx-1 affinity. Mutation of residues 59 (Asp in mouse, Glu in rat epsilon) and 115 (Tyr in mouse, Ser in rat epsilon) converts Wtx-1 affinity for the alpha-epsilon interface of one species to that of the other species. Studies of different mutations at position 59 indicate both steric and electrostatic contributions to Wtx-1 affinity, whereas at position 115, both aromatic and polar groups contribute to affinity. The human nAChR also has lower affinity for Wtx-1 than mouse nAChR, but unlike rat nAChR, residues in both alpha and epsilon subunits mediate the affinity difference. In human nAChR, polar residues (Ser-187 and Thr-189) confer low affinity, whereas in mouse nAChR aromatic residues (Trp-187 and Phe-189) confer high affinity. The overall results show that non-conserved residues at the nAChR binding site, although not crucial for activation by ACh, govern the potency of neuromuscular toxins.     

Subunit interface selectivity of the alpha-neurotoxins for the nicotinic acetylcholine receptor

Peptide toxins selective for particular subunit interfaces of the nicotinic acetylcholine receptor have proven invaluable in assigning candidate residues located in the two binding sites and for determining probable orientations of the bound peptide. We report here on a short alpha-neurotoxin from Naja mossambica mossambica (NmmI) that, similar to other alpha-neurotoxins, binds with high affinity to alphagamma and alphadelta subunit interfaces (KD approximately 100 pM) but binds with markedly reduced affinity to the alphaepsilon interface (KD approximately 100 nM). By constructing chimeras composed of portions of the gamma and epsilon subunits and coexpressing them with wild type alpha, beta, and delta subunits in HEK 293 cells, we identify a region of the subunit sequence responsible for the difference in affinity. Within this region, gammaPro-175 and gammaGlu-176 confer high affinity, whereas Thr and Ala, found at homologous positions in epsilon, confer low affinity. To identify an interaction between gammaGlu-176 and residues in NmmI, we have examined cationic residues in the central loop of the toxin and measured binding of mutant toxin-receptor combinations. The data show strong pairwise interactions or coupling between gammaGlu-176 and Lys-27 of NmmI and progressively weaker interactions with Arg-33 and Arg-36 in loop II of this three-loop toxin. Thus, loop II of NmmI, and in particular the face of this loop closest to loop III, appears to come into close apposition with Glu-176 of the gamma subunit surface of the binding site interface.     

Essential role of B-helix calcium binding sites in annexin V-membrane binding

Crystal structures of annexin V have shown up to 10 bound calcium ions in three different types of binding sites, but previous work concluded that only one of these sites accounted for nearly all of the membrane binding affinity of the molecule. In this study we mutated residues contributing to potential calcium binding sites in the AB and B helices in each of the four domains (eight sites in total) and in DE helices in the first, second, and third domains (three sites in total). We measured the affinity of each protein for phospholipid vesicles and cell membranes by quantitative calcium titration under low occupancy conditions (< 1% saturation of available membrane binding sites). Affinity was calculated from the midpoint and slope of the calcium titration curve and the concentration of membrane binding sites. The results showed that all four AB sites were essential for high affinity binding, as were three of the four B sites (in domains 1, 2, and 3); the DE site in the first domain made a slight contribution to affinity. Multisite mutants showed that each domain contributed additively and independently to binding affinity; in contrast, AB and B sites within the same domain were interdependent. The number of functionally important sites identified was consistent with the Hill coefficient observed in calcium titrations. This study shows an essential and previously unappreciated role for B-helix calcium binding sites in the membrane binding of annexins and indicates that all four domains of the molecule are required for maximum membrane binding affinity.     

Identification of regions within the third FnIII-like domain of the IL-5Ralpha involved in IL-5 interaction

Previously, two binding sites for interleukin 5 (IL-5) were identified on the IL-5 receptor alpha chain (IL-5Ralpha). They are located within the CD loop of the first fibronectin type III (FnIII)-like domain and the EF loop of the second FnIII-like domain. The first binding site was identified by exploiting the different abilities of human IL-5Ralpha (hIL-5Ralpha) and mouse IL-5Ralpha (mIL-5Ralpha) to bind hIL-5. Here we show that ovine IL-5 (oIL-5) has the ability to activate the hIL-5Ralpha but not the mIL-5Ralpha. By using chimeras of the mIL-5Ralpha and hIL-5Ralpha we demonstrate that residues within the first and third FnIII-like domains of mIL-5Ralpha are responsible for this lack of activity. Furthermore, mutation of residues on hIL-5Ralpha to mIL-5Ralpha within the predicted DE and FG loop regions of the third FnIII domain reduces oIL-5 activity. These results show that regions of the third FnIII domain of IL-5Ralpha are involved in binding, in addition to the regions in domains one and two of the IL-5Ralpha that were identified in an earlier study.     

The binding sites for competitive antagonistic, allosteric antagonistic, and agonistic antibodies to the I domain of integrin LFA-1

We explore the binding sites for mAbs to the alpha I domain of the integrin alphaLbeta2 that can competitively inhibit, allosterically inhibit, or activate binding to the ligand ICAM-1. Ten mAbs, some of them clinically important, were mapped to species-specific residues. The results are interpreted with independent structures of the alphaL I domain determined in seven different crystal lattices and in solution, and which are present in three conformational states that differ in affinity for ligand. Six mAbs bind to adjacent regions of the beta1-alpha1 and alpha3-alpha4 loops, which show only small (mean, 0.8 angstroms; maximum, 1.8 angstroms) displacements among the eight I domain structures. Proximity to the ligand binding site and to noncontacting portions of the ICAM-1 molecule explains competitive inhibition by these mAbs. Three mAbs bind to a segment of seven residues in the beta5-alpha6 loop and alpha6 helix, in similar proximity to the ligand binding site, but on the side opposite from the beta1-alpha1/alpha3-alpha4 epitopes, and far from noncontacting portions of ICAM-1. These residues show large displacements among the eight structures in response to lattice contacts (mean, 3.6 angstroms; maximum, 9.4 angstroms), and movement of a buried Phe in the beta5-alpha6 loop is partially correlated with affinity change at the ligand binding site. Together with a lack of proximity to noncontacting portions of ICAM-1, these observations explain variation among this group of mAbs, which can either act as competitive or allosteric antagonists. One agonistic mAb binds distant from the ligand binding site of the I domain, to residues that show little movement (mean, 0.5 angstroms; maximum, 1.0 angstroms). Agonism by this mAb is thus likely to result from altering the orientation of the I domain with respect to other domains within an intact integrin alphaLbeta2 heterodimer.     

Genetic analysis of the first and third extracellular loops of the C5a receptor reveals an essential WXFG motif in the first loop

The extracellular loops of G protein-coupled receptors (GPCRs) frequently contain binding sites for peptide ligands. However, the mechanism of receptor activation following ligand binding and the influence of the extracellular loops in other aspects of receptor function are poorly understood. Here we report a structure-function analysis of the first and third extracellular loops of the human C5a receptor, a GPCR that binds a 74-amino acid peptide ligand. Amino acid substitutions were randomly incorporated into each loop, and functional receptors were identified in yeast. The first extracellular loop contains a large number of positions that cannot tolerate amino acid substitutions, especially residues within the WXFG motif found in many rhodopsin-like GPCRs, yet disruption of these residues does not alter C5a binding affinity. These results demonstrate an unanticipated role for the first extracellular loop, and the WXFG motif in particular, in ligand-mediated activation of the C5a receptor. This motif likely serves a similar role in other GPCRs. The third extracellular loop, in contrast, contains far fewer preserved residues and appears to play a less essential role in receptor activation.     

Detailed analysis of the IL-5-IL-5R alpha interaction: characterization of crucial residues on the ligand and the receptor.

The receptor for interleukin-5 (IL-5) is composed of two different subunits. The IL-5 receptor alpha (IL-5R alpha) is required for ligand-specific binding while association with the beta-chain results in increased binding affinity. Murine IL-5 (mIL-5) has similar activity on human and murine cells, whereas human IL-5 (hIL-5) has marginal activity on murine cells. We found that the combined substitution of K84 and N108 on hIL-5 by their respective murine counterpart yields a molecule which is as potent as mIL-5 for growth stimulation of a murine cell line. Since the unidirectional species specificity is due only to the interaction with the IL-5R alpha subunit, we have used chimeric IL-5R alpha molecules to define regions of hIL-5R alpha involved in species-specific hIL-5 ligand binding. We found that this property is largely determined by the NH2-terminal module of hIL-5R alpha, and detailed analysis defined D56 and to a lesser extent E58 as important for binding. Moreover, two additional residues, D55 and Y57, were identified by alanine scanning mutagenesis within the same region. Based on the observed homology between the NH2-terminal module and the membrane proximal (WSXWS-containing) module of hIL-5R alpha we located this stretch of four amino acid residues (D55, D56, Y57 and E58) in the loop region that connects the C and D beta-strands on the proposed tertiary structure of the NH2-terminal module.(ABSTRACT TRUNCATED AT 250 WORDS)     

High affinity ligand binding is not essential for granulocyte-macrophage colony-stimulating factor receptor activation.

The high affinity receptor of the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is a heterodimer composed of two members of the cytokine receptor superfamily. GM-CSF binds to the alpha-subunit (GM-R alpha) with low affinity and to the receptor alpha beta complex (GM-R alpha beta) with high affinity. The GM-CSF.GM-R alpha beta complex is responsible for biological activity. Interactions of the N-terminal helix of mouse GM-CSF with mGM-R alpha beta were examined by introducing single alanine substitutions of hydrophilic residues in this region of mGM-CSF. The consequences of these substitutions were evaluated by receptor binding and biological assays. Although all mutant proteins exhibited near wild-type biological activity, most were defective in high affinity receptor binding. In particular, substitution of Glu-21 with alanine abrogated high affinity binding leaving low affinity binding unaffected. Despite near wild-type biological activity, no detectable binding interaction of this mutant with mGM-R beta in the context of mGM-R alpha beta was observed. Cross-linking studies showed an apparent interaction of this mutant protein with mGM-R alpha beta. The deficient receptor binding characteristics and near wild-type biological activity of this mutant protein demonstrate that mGM-CSF receptor activation can occur independently of high affinity binding, suggesting that conformational changes in the receptor induced by mGM-CSF binding generate an active ligand-receptor complex.     

The role of the N-terminal domain of the complement fragment receptor C5L2 in ligand binding

C5L2 is a new cellular receptor found to interact with the human anaphylatoxins complement factor C5a and its C-terminal cleavage product C5a des Arg. The classical human C5a receptor (C5aR) preferentially binds C5a, with a 10-100-fold lower affinity for C5a des Arg. In contrast, C5L2 binds both ligands with nearly equal affinity. C5aR presents acidic and tyrosine residues in its N terminus that interact with the core of C5a while a hydrophobic pocket formed by the transmembrane helices interacts with residues in the C terminus of C5a. Here, we have investigated the molecular basis for the increased affinity of C5L2 for C5a des Arg. Rat and mouse C5L2 preferentially bound C5a des Arg, whereas rodent C5aR showed much higher affinity for intact C5a. Effective peptidic and non-peptidic ligands for the transmembrane hydrophobic pocket of C5aR were poor inhibitors of ligand binding to C5L2. An antibody raised against the N terminus of human C5L2 did not affect the binding of C5a to C5L2 but did inhibit C5a des Arg binding. A chimeric C5L2, containing the N terminus of C5aR, had little effect on the affinity for C5a des Arg. Mutation of acidic and tyrosine residues in the N terminus of human C5L2 revealed that 3 residues were critical for C5a des Arg binding but had little involvement in C5a binding. C5L2 thus appears to bind C5a and C5a des Arg by different mechanisms, and, unlike C5aR, C5L2 uses critical residues in its N-terminal domain for binding only to C5a des Arg.     

A simple method for production and purification of soluble and biologically active recombinant human leukemia inhibitory factor (hLIF) fusion protein in Escherichia coli

Mouse embryonic stem cells (mESCs) rely on a cytokine named leukemia inhibitory factor (LIF) to maintain their undifferentiated state and pluripotency. However, the progress of mESC research is restricted and limited to highly funded laboratories due to the cost of commercial LIF. Here we presented the homemade hLIF which is biologically active. The hLIF cDNA was cloned into two different vectors in order to produce N-terminal His₆-tag and Trx-His₆-tag hLIF fusion proteins in Origami(DE3) Escherichia coli. The His₆-hLIF fusion protein was not as soluble as the Trx-His₆-hLIF fusion protein. One-step immobilized metal affinity chromatography (IMAC) was done to recover high purity (>95% pure) His₆-hLIF and Trx-His₆-hLIF fusion proteins with the yields of 100 and 200 mg/l of cell culture, respectively. The hLIF fusion proteins were identified by Western blot and verified by mass spectrometry (LC/MS/MS). The hLIF fusion proteins specifically promote the proliferation of TF-1 cells in a dose-dependent manner. They also demonstrate the potency to retain the morphology of undifferentiated mESCs, in that they were positive for mESC markers (Oct-4, Sox-2, Nanog, SSEA-1 and alkaline phosphatase activity). These results demonstrated that the N-terminal fusion tags of the His₆-hLIF and Trx-His₆-hLIF fusion proteins do not interfere with their biological activity. This expression and purification approach to produce recombinant hLIF is a simple, reliable, cost effective and user-friendly method.     

Structure and function of the regulatory HRDC domain from human Bloom syndrome protein

The helicase and RNaseD C-terminal (HRDC) domain, conserved among members of the RecQ helicase family, regulates helicase activity by virtue of variations in its surface residues. The HRDC domain of Bloom syndrome protein (BLM) is known as a critical determinant of the dissolution function of double Holliday junctions by the BLM–Topoisomerase IIIα complex. In this study, we determined the solution structure of the human BLM HRDC domain and characterized its DNA-binding activity. The BLM HRDC domain consists of five α-helices with a hydrophobic 3₁₀-helical loop between helices 1 and 2 and an extended acidic surface comprising residues in helices 3–5. The BLM HRDC domain preferentially binds to ssDNA, though with a markedly low binding affinity (K_d ∼100 μM). NMR chemical shift perturbation studies suggested that the critical DNA-binding residues of the BLM HRDC domain are located in the hydrophobic loop and the N-terminus of helix 2. Interestingly, the isolated BLM HRDC domain had quite different DNA-binding modes between ssDNA and Holliday junctions in electrophoretic mobility shift assay experiments. Based on its surface charge separation and DNA-binding properties, we suggest that the HRDC domain of BLM may be adapted for a unique function among RecQ helicases—that of bridging protein and DNA interactions.