System For Production Of Recombinant Proteins Research Articles

Recombinant protein expression in Pichia pastoris.

The methylotrophic yeast Pichia pastoris is now one of the standard tools used in molecular biology for the generation of recombinant protein. P. pastoris has demonstrated its most powerful success as a large-scale (fermentation) recombinant protein production tool. What began more than 20 years ago as a program to convert abundant methanol to a protein source for animal feed has been developed into what is today two important biological tools: a model eukaryote used in cell biology research and a recombinant protein production system. To date well over 200 heterologous proteins have been expressed in P. pastoris. Significant advances in the development of new strains and vectors, improved techniques, and the commercial availability of these tools coupled with a better understanding of the biology of Pichia species have led to this microbe's value and power in commercial and research labs alike.

Production of recombinant proteins by methylotrophic yeasts

The methylotrophic yeasts Hansenula polymorpha, Pichia pastoris and Candida boidinii have been developed as production systems for recombinant proteins. The favourable and most advantageous characteristics of these species have resulted in an increasing number of biotechnological applications. As a consequence, these species — especially H. polymorpha and P. pastoris — are rapidly becoming the systems of choice for heterologous gene expression in yeast. Recent advances in the development of these yeasts as hosts for the production of heterologous proteins have provided a catalogue of new applications, methods and system components.

Efficient and inducible production of human interleukin 6 in Chinese hamster ovary cells using a novel expression system.

High level and inducible production of human interleukin 6 (hIL-6) was achieved using a novel expression system in Chinese hamster ovary (CHO) cells. In this system, the transcription of hIL-6 gene under the control of PhCMV*-1 promoter composed of tetracycline operator sequences and a minimal promoter is activated by a chimeric transactivator (tTA) composed of tetracycline repressor and transactivating domain of VP16 protein of herpes simplex virus. The transcription of tTA gene, which is also under the control of PhCMV*-1 promoter, is activated by itself via a positive feedback cycle. The expression of both genes is further enhanced by potentiating the VP16 transactivating domain of tTA transactivator with pX protein of hepatitis B virus. In the presence of tetracycline, the tTA transactivators can not bind to PhCMV*-1 promoter, therefore, the expression of hIL-6 and tTA gene is suppressed, and the pX will not activate basal transcription. In the absence of tetracycline, tTA transactivators bind to PhCMV*-1 promoter and activate efficient transcription of hIL-6 and tTA gene, and the transcription is further enhanced by pX via VP16 transactivating domain. Using this strategy, we isolated a clone (UX1) producing hIL-6 at a rate about 1425 ng/10(6) cells/day. Furthermore, the hIL-6 production is stringently regulated by tetracycline. This results suggested a novel strategy to establish highly efficient, inducible and cell type independent recombinant protein production system by using an artificial promoter to recruit transactivators and coactivators which can synergistically activate transcription.

A novel promoter, derived from the isocitrate lyase gene of Candida tropicalis, inducible with acetate in Saccharomyces cerevisiae

When the isocitrate lyase gene, containing 5'-upstream and 3'-flanking regions, of an n-alkane-assimilating yeast Candida tropicalis was introduced into Saccharomyces cerevisiae, the enzyme was functionally overexpressed in the cells grown on acetate. The amount of the recombinant isocitrate lyase expressed in S. cerevisiae was as much as 30% of the total soluble proteins in the cells, being comparable to that with GAL7 functional under the control of galactose. The expression was also observed when the cells were grown on glycerol, lactate, ethanol or oleate. These facts indicate that the isocitrate lyase gene upstream region (UPR-ICL) contains a strong promoter functional in S. cerevisiae. UPR-ICL is active as a promoter on cheap carbon sources such as acetate and nonconventional carbon sources such as oleate, whereas many conventional strong promoters demand relatively expensive sugars or sugar derivatives. Therefore, it is promising to construct an economical recombinant protein production system by using UPR-ICL.

Product deactivation in recombinant Streptomyces

The production of tyrosinase by Streptomyces antibioticus (p1J7O2) was investigated as a model system for recombinant protein production by Streptomyces. Product deactivation was found to have a severe effect on the levels of tyrosinase obtained. Tyrosinase deactivation was detected during all phases of batch cultures, with higher specific deactivation rates observed during the stationary phase. The specific deactivation rate exhibited an Arrhenius dependence on temperature, with approximately a twofold increase in the deactivation rate between 25 degrees C and 30 degrees C. The effect of deactivation on the determination of tyrosinase production kinetics is discussed. A strategy was implemented to increase tyrosinase productivity by enriching the growth medium and reducing the culture temperature during the period of maximum tyrosinase production. This strategy resulted in a shorter culture time and a 2.5-fold increase in tyrosinase activity compared to a culture grown at 25 degrees C using a standard growth medium.