Stable, high yield expression of gp145 Env glycoprotein from HIV-1 in mammalian cells.

The HIV-1 derived gp145 protein is being investigated by research groups as preclinical studies have shown high promise for this protein as a vaccine against HIV. However, one of the main challenges with manufacturing this promising protein has been ascribed to the low yield obtained in mammalian cell cultures. Significant improvements in gp145 production are needed to address this issue to test the gp145 protein as a potentially effective, safe, and affordable HIV vaccine. Here we describe the application of a novel expression technology to create GMP-grade CHO cell lines expressing approximately 50 µg/ml in non-optimized fed-batch culture, which is an order of magnitude higher than that obtained in existing processes. Top producing clones show a high degree of similarity in the glycosylation patterns of the purified protein to the reference standard. Conformational integrity and functionality was demonstrated via high-affinity binding to soluble CD4, using a panel of antibodies including VRC01, F105, Hk20, PG9 and 17b. In summary, we were able to generate CHO cell lines expressing HIV gp145 with significantly higher overall expression yields than currently accessible, and high product quality that could potentially be suitable for future studies assessing the efficacy and safety of gp145-based HIV vaccines.

STEP® vectors for rapid generation of stable transfected CHO cell pools and clones with high expression levels and product quality homogeneity of difficult-to-express proteins.

Many proteins produced in CHO cells need evaluation for their clinical and commercial potential. Traditional methods based on stable clone generation are slow and unsuitable for screening larger numbers of proteins, while transient expression technologies are fast but unpredictable regarding product quality and lacking an optional path to subcloning. The STEP® vector technology introduced here combines the best properties of both methods. STEP® vectors contain a strong transcriptional cassette driving expression of a bicistronic mRNA. The gene-of-interest (GOI) is cloned upstream of a functionally impaired zeocin resistance gene (FI-Zeo) whose translation is coupled to that of the GOI through an IRES. Stable transfected cells surviving zeocin selection produce high levels of FI-Zeo and thus, high levels of the GOI-encoded protein. By using different spacers, the translational coupling efficiency and selection strength can be controlled allowing maximization of expression of any GOI. Production of laronidase and factor VII (FVII) is presented as examples of unrelated, difficult-to-express (DTE) proteins. First step is rapid generation of transfected pools with the STEP® vectors. All high expressing surviving pools showed high product quality homogeneity as did monoclonal cell lines obtained from the top pools. Up to 500 µg/mL laronidase was obtained with virtually identical glycosylation profile as reference product. For FVII, cell specific productivity of 0.45 pg/cell/day with 50 IU/µg protein matched highest reported levels of reference product even before process development. Taken together, STEP® vector technology is ideally suited for rapid, small to large-scale production of DTE proteins compared to traditional methods.

Structure-guided Discovery of Dual-recognition Chemibodies.

Small molecules and antibodies each have advantages and limitations as therapeutics. Here, we present for the first time to our knowledge, the structure-guided design of "chemibodies" as small molecule-antibody hybrids that offer dual recognition of a single target by both a small molecule and an antibody, using DPP-IV enzyme as a proof of concept study. Biochemical characterization demonstrates that the chemibodies present superior DPP-IV inhibition compared to either small molecule or antibody component alone. We validated our design by successfully solving a co-crystal structure of a chemibody in complex with DPP-IV, confirming specific binding of the small molecule portion at the interior catalytic site and the Fab portion at the protein surface. The discovery of chemibodies presents considerable potential for novel therapeutics that harness the power of both small molecule and antibody modalities to achieve superior specificity, potency, and pharmacokinetic properties.

Kinetic solvent isotope effect in steady-state turnover by CYP19A1 suggests involvement of Compound 1 for both hydroxylation and aromatization steps.

CYP19A1, or human aromatase catalyzes the conversion of androgens to estrogens in a three-step reaction through the formation of 19-hydroxy and 19-aldehyde intermediates. While the first two steps of hydroxylation are thought to proceed through a high-valent iron-oxo species, controversy exists surrounding the identity of the reaction intermediate that catalyzes the lyase and aromatization reaction. We investigated the kinetic isotope effect on the steady-state turnover of Nanodisc-incorporated human CYP19A1 to explore the mechanisms of this reaction. Our experiments reveal a significant (∼ 2.5) kinetic solvent isotope effect for the C10-C19 lyase reaction, similar to that of the first two hydroxylation steps (2.7 and 1.2). These data implicate the involvement of Compound 1 as a reactive intermediate in the final aromatization step of CYP19A1.

Resonance Raman spectroscopy of the oxygenated intermediates of human CYP19A1 implicates a compound i intermediate in the final lyase step.

CYP19A1, or aromatase, a cytochrome P450 responsible for estrogen biosynthesis in humans, is an important therapeutic target for the treatment of breast cancer. There is still controversy surrounding the identity of reaction intermediate that catalyzes carbon-carbon scission in this key enzyme. Probing the oxy-complexes of CYP19A1 poised for hydroxylase and lyase chemistries using resonance Raman spectroscopy and drawing a comparison with CYP17A1, we have found no significant difference in the frequencies or isotopic shifts for these two steps in CYP19A1. Our experiments implicate the involvement of Compound I in the terminal lyase step of CYP19A1 catalysis.

Temperature derivative spectroscopy to monitor the autoxidation decay of cytochromes P450.

Temperature derivative spectroscopy (TDS), a type of relaxation spectroscopy, is a powerful tool to study protein dynamics (Berendzen, J.; Braunstein, D. Proc. Natl. Acad. Sci. U. S. A. 1990, 87, 1). We developed the version of temperature derivative spectroscopy to monitor kinetics of autoxidation of cytochromes P450 and applied it to study the properties of the oxy-ferrous complex of a human membrane bound P450, CYP19A1 (aromatase), and that of a bacterial soluble P450, CYP101 when bound with their most common substrates, androstenedione (AD) and camphor, respectively. TDS extends the panel of methods that can be used to monitor heme protein kinetics, providing a rapid measurement technique and enabling measurement of the autoxidation rate over a wide range of temperatures, yielding the activation energy as well as absolute reaction rate in a single experiment.

Spectroscopic features of cytochrome P450 reaction intermediates.

Cytochromes P450 constitute a broad class of heme monooxygenase enzymes with more than 11,500 isozymes which have been identified in organisms from all biological kingdoms [1]. These enzymes are responsible for catalyzing dozens chemical oxidative transformations such as hydroxylation, epoxidation, N-demethylation, etc., with very broad range of substrates [2,3]. Historically these enzymes received their name from 'pigment 450' due to the unusual position of the Soret band in UV-vis absorption spectra of the reduced CO-saturated state [4,5]. Despite detailed biochemical characterization of many isozymes, as well as later discoveries of other 'P450-like heme enzymes' such as nitric oxide synthase and chloroperoxidase, the phenomenological term 'cytochrome P450' is still commonly used as indicating an essential spectroscopic feature of the functionally active protein which is now known to be due to the presence of a thiolate ligand to the heme iron [6]. Heme proteins with an imidazole ligand such as myoglobin and hemoglobin as well as an inactive form of P450 are characterized by Soret maxima at 420nm [7]. This historical perspective highlights the importance of spectroscopic methods for biochemical studies in general, and especially for heme enzymes, where the presence of the heme iron and porphyrin macrocycle provides rich variety of specific spectroscopic markers available for monitoring chemical transformations and transitions between active intermediates of catalytic cycle.

A method for computing the inter-residue interaction potentials for reduced amino acid alphabet.

Inter-residue potentials are extensively used in the design and evaluation of protein structures. However,dealing with all (20 x 20) interactions becomes computationally difficult in extensive investigations. Hence, it is desirable to reduce the alphabet of 20 amino acids to a smaller number. Currently, several methods of reducing the residue types exist; however a critical assessment of these methods is not available. Towards this goal,here we review and evaluate different methods by comparing with the complete (20 x 20) matrix of Miyazawa-Jernigan potential, including a method of grouping adopted by us, based on multi dimensional scaling (MDS). The second goal of this paper is the computation of inter-residue interaction energies for the reduced amino acid alphabet, which has not been explicitly addressed in the literature until now. By using a least squares technique, we present a systematic method of obtaining the interaction energy values for any type of grouping scheme that reduces the amino acid alphabet. This can be valuable in designing the protein structures.