Marion Mahnke

Biochemical characterization of USP7 reveals post‐translational modification sites and structural requirements for substrate processing and subcellular localization

Ubiquitin specific protease 7 (USP7) belongs to the family of deubiquitinating enzymes. Among other functions, USP7 is involved in the regulation of stress response pathways, epigenetic silencing and the progress of infections by DNA viruses. USP7 is a 130‐kDa protein with a cysteine peptidase core, N‐ and C‐terminal domains required for protein–protein interactions. In the present study, recombinant USP7 full length, along with several variants corresponding to domain deletions, were expressed in different hosts in order to analyze post‐translational modifications, oligomerization state, enzymatic properties and subcellular localization patterns of the enzyme. USP7 is phosphorylated at S18 and S963, and ubiquitinated at K869 in mammalian cells. In in vitro activity assays, N‐ and C‐terminal truncations affected the catalytic efficiency of the enzyme different. Both the protease core alone and in combination with the N‐terminal domain are over 100‐fold less active than the full length enzyme, whereas a construct including the C‐terminal region displays a rather small decrease in catalytic efficiency. Limited proteolysis experiments revealed that USP7 variants containing the C‐terminal domain interact more tightly with ubiquitin. Besides playing an important role in substrate recognition and processing, this region might be involved in enzyme dimerization. USP7 constructs lacking the N‐terminal domain failed to localize in the cell nucleus, but no nuclear localization signal could be mapped within the enzymes first 70 amino acids. Instead, the tumor necrosis factor receptor associated factor‐like region (amino acids 70–205) was sufficient to achieve the nuclear localization of the enzyme, suggesting that interaction partners might be required for USP7 nuclear import.

Automated baculovirus titration assay based on viable cell growth monitoring using a colorimetric indicator

The fastest methods reported are based on viral DNA quantitation using either flow cytometry (10) or real-time PCR (11), which allow titer determination within two hours. The drawbacks of these approaches are the costs of equipment and staining reagents, and the fact that the total number of particles and not the number of infectious particles is determined. An alternative approach is based on the lytic nature of the viral system (12), and more specifically on the fact that cell growth is attenuated upon virus infection. This growth reduction is dose-dependent and can be estimated by measuring the viable cell concentration and subsequently correlating this to the virus titer. Indeed, a new method was recently developed for virus titration by spectrophotometrically monitoring the cell viability with 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide (MTT) (4). The accuracy of the method was clearly demonstrated; however, the number of preparation steps and the overall duration (6 days) are not compatible with a fast and automated high-throughput process. In this work, we demonstrate that

High-Throughput Insect Cell Protein Expression Applications

The Baculovirus Expression Vector System (BEVS) is one of the most efficient systems for production of recombinant proteins and consequently its application is wide-spread in industry as well as in academia. Since the early 1970s, when the first stable insect cell lines were established and the infectivity of bacu-lovirus in an in vitro culture system was demonstrated (1, 2), virtually thousands of reports have been published on the successful expression of proteins using this system as well as on method improvement. However, despite its popularity the system is labor intensive and time consuming. Moreover, adaptation of the system to multi-parallel (high-throughput) expression is much more difficult to achieve than with E. coli due to its far more complex nature. However, recent years have seen the development of strategies that have greatly enhanced the stream-lining and speed of baculovirus protein expression for increased throughput via use of automation and miniaturization. This chapter therefore tries to collate these developments in a series of protocols (which are modifications to standard procedure plus several new approaches) that will allow the user to expedite the speed and throughput of baculovirus-mediated protein expression and facilitate true multi-parallel, high-throughput protein expression profiling in insect cells. In addition we also provide a series of optimized protocols for small and large-scale transient insect cell expression that allow for both the rapid analysis of multiple constructs and the concomitant scale-up of those selected for on-going analysis. Since this approach is independent of viral propagation, the timelines for this approach are markedly shorter and offer a significant advantage over standard bacu-lovirus expression approach strategies in the context of HT applications.

Cloning and pharmacological characterization of CCR7, CCL21 and CCL19 from Macaca fascicularis

The chemokine receptor CCR7 and its ligands CCL19 and CCL21 play an important role in lymphocyte homing and have also been associated with inflammatory, allergic and lung disorders. Cloning of the cynomolgus monkey genes encoding CCR7, CCL19 and CCL21 revealed 93-97% sequence identity of the deduced proteins with their respective human homologs. In chemotaxis assays, B300-19 cells transfected with the cynomolgus (c) CCR7 receptor migrated in response to cCCL19 and cCCL21 in a dose-dependent manner with EC(50) values of 324+/-188nM and 247+/-29nM, respectively. cCCL19 and cCCL21 also elicited calcium responses in stable cell CHO-K1 lines expressing the cCCR7 receptor with EC(50) values of 227+/-4nM and 484+/-163nM, respectively. Although both human (h) CCL19 and hCCL21 elicited increases in intracellular calcium at the cCCR7 receptor, hCCL19 almost completely inhibited subsequent stimulation by hCCL21 whilst hCCL21 failed to inhibit subsequent stimulation by hCCL19. These results identify novel cynomolgus monkey genes and provide a model system for pre-clinical studies of potential drug candidates.

Efficient Processes for Protein Expression Using Recombinant Baculovirus Particles

The use of baculoviruses has become a standard approach in many labs for recombinant protein production. In addition to giving a broad and practical overview of the technology, this chapter focuses in particular on two recent developments in the field and how these can be efficiently exploited for protein production: the use of baculovirus-infected insect cells and in vivo recombination-mediated production of recombinant viruses.

Optimization of the anti-(human CD3) immunotoxin DT389-scFv(UCHT1) N-terminal sequence to yield a homogeneous protein

The production and regulatory approval processes for biopharmaceuticals require detailed characterization of potential products. Therapeutic proteins should preferably be homogeneous, although limited, reproducible, heterogeneity may be tolerated. A diphtheria toxin‐based anti‐(human CD3) immunotoxin, DT389–scFv(UCHT1), was expressed in Escherichia coli and purified following refolding [DT389 corresponds to amino acids 1–389 of diphtheria toxin, scFv is single‐chain variable‐region antibody fragment and UCHT1is an anti‐(human CD3) monoclonal antibody]. Biochemical characterization of this molecule by MS and N‐terminal sequencing by Edman degradation revealed that the protein was heterogeneous at the N‐terminus, containing species both with (60%) and without (40%) the initiator methionine residue. In an attempt to generate an N‐terminally homogeneous molecule, a panel of seven N‐terminal variants was designed, based on the published specificity of bacterial methionine aminopeptidase. Following bacterial expression, partial purification and separation on SDS/PAGE, these proteins were subjected to N‐terminal sequencing by Edman degradation. Three of the mutants yielded a 100% homogeneous amino acid sequence. By contrast, the original DT389–scFv(UCHT1) protein and four variant proteins yielded two sequences with varying ratios corresponding to species with and without methionine. The N‐terminal sequences of the three homogeneous clones were MLADD and MLDD, where the methionine was completely retained, and SADD, where the methionine was completely removed. One of the homogeneous mutants (SADD) was expressed, refolded and purified and found to be equipotent with the parent immunotoxin. Thus, using a rational mutagenesis approach, three N‐terminally homogeneous variants of DT389–scFv(UCHT1) have been identified, at least one of which is functionally indistinguishable from the parent immunotoxin. This approach is generally applicable to biopharmaceutical production and immunotoxin development in particular.

Automation for higher throughput in protein expression: visions, facts and fictions

Down-scaling, parallelization and automation are newtrends in the field of recombinant protein expression inthe post genomic era [1-3]. During the past years manycompanies and academic institutions have heavilyinvested in process and automation technologies. Doesthis trend keep its promise? Can post genomic proteinproduction issues be overcome with few automated proc-esses?This abstract wants to highlight two years of experience inrunning a Protein Production Center in an industrial envi-ronment applying the expression systems BEVS,