Current Pharmaceutical Biotechnology - Volume 11, Issue 3, 2010

Biotechnology industries commenced with the production of recombinant proteins for developing biopharmaceuticals. However, scientific development of recombinant technologies has contributed not only to protein pharmaceuticals but also to industrial enzymes, drug development and material sciences. Since the beginning of recombinant technology, numerous pharmaceutical proteins have been developed, coveri Read More

Mammalian cell lines are important host cells for the industrial production of pharmaceutical proteins owing to their capacity for correct folding, assembly and post-translational modification. In particular, Chinese hamster ovary (CHO) cells are the most dependable host cells for the industrial production of therapeutic proteins. Growing demand for therapeutic proteins promotes the development of technologies for high quality Read More

The expression of proteins which do not express well on their own can be enhanced by linking them to human serum albumin (HSA) or antibody crystallizable fragment (Fc). The constructs shown here are designed to secrete the proteins after transient transfection of mammalian cell lines. The fusion partners are appended to the N-terminus of the proteins and contain a linker designed to be proteolytically cleaved. Transient Read More

While the Baculovirus Expression Vector System (BEVS) mainly uses insect cell lines, such as Sf9 cells, the robust high expression system using silkworm has also been developed. We have further improved technologies for enhancement of virus recombination, reduction of proteolytic degradation and aggregation, and more reliable promoters. These developments made it possible to achieve high and soluble expressio Read More

Brevibacillus expression system is an effective bacterial expression system for secretory proteins. The host bacterium, Brevibacillus choshinensis, a gram-positive bacterium, has strong capacity to secrete a large amount of proteins (∼30g/L), which mostly consist of cell wall protein. A host-vector system that utilizes such high expression capacity has been constructed for the production of secretory proteins and tested for various h Read More

A novel expression of recombinant proteins was developed using moderate halophiles that accumulate osmolytes and hence provide cytoplasmic environments where osmolyte-driven folding can take place. Promoters and selection marker were developed for high expression of foreign proteins. Examples are given for expression of bacterial nucleoside diphosphate kinase and human serine racemase.

The PURE system is a highly controllable cell-free protein synthesis system composed of individually prepared components that are required for protein synthesis in Escherichia coli. The PURE system contains neither nucleases nor proteases, both of which degrade DNA or mRNA templates and proteins. The protein products are easily purified using affinity chromatography to remove the tagged protein factors. The PURE sys Read More

We have made a dramatic improvement of the wheat cell-free protein synthesis system. The first key improvement is the method for preparation of the cell-free extract that is free of inhibitory factors of translation reaction. Additional improvements include a method for preparation of transcription-ready templates by PCR, an expression vector for the cell-free system, and the “bilayer” mode reaction method that is mu Read More

Cell-free protein synthesis systems offer production of native proteins with high speed, even for the proteins that are toxic to cells. Among cell-free systems, the system derived from insect cells has the potential to carry out posttranslational modifications that are specific to eukaryotic organisms, as occurs in the rabbit reticulocyte system. In this review, we describe development of this insect cell-free system and its applications.

Protein refolding is still on trial-and-error basis. Here we describe step-wise dialysis refolding, in which denaturant concentration is altered in step-wise fashion. This technology controls the folding pathway by adjusting the concentrations of the denaturant and other solvent additives to induce sequential folding or disulfide formation.

Protein refolding using urea gradient size exclusion chromatography (UGSEC) is a new version of conventional SEC refolding, which incorporates urea gradient into the SEC. Operating factors, mainly the urea gradient length and the final urea concentration in the gradient, are discussed in detail. Recent applications of UGSEC, including refolding of recombinant human granulocyte colony stimulating factor by UGSEC, is show Read More

We have developed a high-pH, pH-shift refolding “Ph-Fold technology”, both for academic research and industrial protein drug development applications. Using this technology, we were able to refold some “difficult-to-refold” proteins, some of which are proven important drug targets and protein drug candidates. The technology is composed of the initial E. coli production of inclusion bodies, the high pH solubilization/pH shif Read More

Molecular chaperones in living systems inspired us to explore new concepts for assisting protein refolding. The chaperone selectively interacts with a non-native protein by hydrophobic interaction to prevent irreversible aggregation and releases the protein in its refolded form with the aid of ATP and another co-chaperone. Cyclodextrins have been used to simulate the function of the chaperones by controlling the hydrophobic inte Read More

Protein refolding from unfolded state is usually carried out at low temperature to reduce protein aggregation and proteolytic degradation. This review briefly introduces a unique method for the protein refolding via high temperature, typically at above melting temperature of the protein.

It has been a conventional notion that cytoplasmic recombinant expression leads to either soluble protein or inclusion bodies. In the latter case, it was always assumed that proteins in inclusion bodies (IBs) are more or less unfolded and hence require complete denaturing condition for solubilization, which uses strong detergents, urea or guanidine hydrochloride. However, we often observe distribution of expressed prot Read More

Aim: To study the effects of an extract solution from radix curcumae on the expressions of VEGF, COX-2 and PCNA in gastric mucosa of rats during carcinogenesis induced MNNG. Methods: Eighty male Wistar rats were randomly divided into 5 groups: group A, water and normal saline; group B, MNNG and normal saline; group C, MNNG and celecoxib; group D, MNNG and low-dose (1g/ml) radix curcumae steam distillation extract Read More