Optimization of insect cell based protein production processes - online monitoring, expression systems, scale-up

Due to the increasing use of insect cell based expression systems in research and industrial recombinant protein production, the development of efficient and reproducible production processes remains a challenging task. In this context, the application of online monitoring techniques is intended to ensure high and reproducible product qualities already during the early phases of process development. In the following chapter, the most common transient and stable insect cell based expression systems are briefly introduced. Novel applications of insect cell based expression systems for the production of insect derived antimicrobial peptides/proteins (AMPs) are discussed using the example of G. mellonella derived gloverin. Suitable in situ sensor techniques for insect cell culture monitoring in disposable and common bioreactor systems are outlined with respect to optical and capacitive sensor concepts. Since scale-up of production processes is one of the most critical steps in process development, a conclusive overview is given about scale up aspects for industrial insect cell culture processes

Silkworm expression system as a platform technology in life science

Many recombinant proteins have been successfully produced in silkworm larvae or pupae and used for academic and industrial purposes. Several recombinant proteins produced by silkworms have already been commercialized. However, construction of a recombinant baculovirus containing a gene of interest requires tedious and troublesome steps and takes a long time (3–6 months). The recent development of a bacmid, Escherichia coli and Bombyx mori shuttle vector, has eliminated the conventional tedious procedures required to identify and isolate recombinant viruses. Several technical improvements, including a cysteine protease or chitinase deletion bacmid and chaperone-assisted expression and coexpression, have led to significantly increased protein yields and reduced costs for large-scale production. Terminal N-acetyl glucosamine and galactose residues were found in the N-glycan structures produced by silkworms, which are different from those generated by insect cells. Genomic elucidation of silkworm has opened a new chapter in utilization of silkworm. Transgenic silkworm technology provides a stable production of recombinant protein. Baculovirus surface display expression is one of the low-cost approaches toward silkworm larvae-derived recombinant subunit vaccines. The expression of pharmaceutically relevant proteins, including cell/viral surface proteins, membrane proteins, and guanine nucleotide-binding protein (G protein) coupled receptors, using silkworm larvae or cocoons has become very attractive. Silkworm biotechnology is an innovative and easy approach to achieve high protein expression levels and is a very promising platform technology in the field of life science. Like the “Silkroad,” we expect that the “Bioroad” from Asia to Europe will be established by the silkworm expression system

Evolutionary Trajectory of White Spot Syndrome Virus (WSSV) Genome Shrinkage during Spread in Asia

Background - White spot syndrome virus (WSSV) is the sole member of the novel Nimaviridae family, and the source of major economic problems in shrimp aquaculture. WSSV appears to have rapidly spread worldwide after the first reported outbreak in the early 1990s. Genomic deletions of various sizes occur at two loci in the WSSV genome, the ORF14/15 and ORF23/24 variable regions, and these have been used as molecular markers to study patterns of viral spread over space and time. We describe the dynamics underlying the process of WSSV genome shrinkage using empirical data and a simple mathematical model. Methodology/Principal Findings - We genotyped new WSSV isolates from five Asian countries, and analyzed this information together with published data. Genome size appears to stabilize over time, and deletion size in the ORF23/24 variable region was significantly related to the time of the first WSSV outbreak in a particular country. Parameter estimates derived from fitting a simple mathematical model of genome shrinkage to the data support a geometric progression (

The Gene Pool Concept Applied to Crop Wild Relatives: An Evolutionary Perspective

Crop wild relatives (CWR) can provide important resources for the genetic improvement of cultivated species. Because crops are often related to many wild species and because exploration of CWR for useful traits can take many years and substantial resources, the categorization of CWR based on a comprehensive assessment of their potential for use is an important knowledge foundation for breeding programs. The initial approach for categorizing CWR was based on crossing studies to empirically establish which species were interfertile with the crop. The foundational concept of distinct gene pools published almost 50 years ago was developed from these observations. However, the task of experimentally assessing all potential CWR proved too vast; therefore, proxies based on phylogenetic and other advanced scientific information have been explored. A current major approach to categorize CWR aims to comprehensively synthesize experimental data, taxonomic information, and phylogenetic studies. This approach very often ends up relying not only on the synthesis of data but also intuition and expert opinion and is therefore difficult to apply widely in a reproducible manner. Here, we explore the potential for a stronger standardization of the categorization method, with focus on evolutionary relationships among species combined with information on patterns of interfertility between species. Evolutionary relationships can be revealed with increasing resolution via next-generation sequencing, through the application of the multispecies coalescent model and using focused analyses on species discovery and delimitation that bridge population genetics and phylogenetics fields. Evolutionary studies of reproductive isolation can inform the understanding of patterns of interfertility in plants. For CWR, prezygotic postpollination reproductive barriers and intrinsic postzygotic barriers are the most important factors and determine the probability of producing viable and fertile offspring. To further the assessment of CWR for use in plant breeding, we present observed and predicted gene pool indices. The observed index quantifies patterns of interfertility based on fertilization success, seed production, offspring viability, and hybrid fertility. The predicted gene pool index requires further development of the understanding of quantitative and qualitative relationships between reproductive barriers, measures of genetic relatedness, and other relevant characteristics for crops and their wild relatives