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Custom Protein Expression Creative
Biolabs has an established track record in
producing recombinant proteins in bacterial, yeast, baculoviral or
mammalian systems in soluble forms of biological activities. We offer a
full range of services from gene synthesis to protein purification. In
order to obtain a recombinant protein with satisfactory function and
yield, experimental protocols are optimized at every step of the protein
expression process to match the requirements of the final protein
application. o Bacterial systems (E. coli. and Bacillus systems) o Traditional E. coli expression systems that employ IPTG induction, various host strains and distinct fusion tags are available; o We also have a high level, high throughput E. coli expression system using auto-induction technology, which enables regulated protein expression in E. coli without the need to monitor the culture or add inducer during cell growth. The yields we have achieved range between 20~100mg/L. o We have another proprietary soluble expression system that expresses recombinant proteins predominantly into cytoplasm in active native forms at high yields. o In addition, we have co-expressed quite a few human protein pairs in e. coli cells using our proprietary bacterial co-expression system. The co-expression system contains two complete [while independent] expression cassettes that are located on one single vector. Of note, using this system, a fusion tag, e.g. HIS, Flag, V5 or S-tag can be easily integrated on each of the two proteins.
o A bacillus expression system is another unique expression
system we have. The bacillus expression system is featured by
its secreted expression. The secreted proteins usually retain its biological
activities. o Yeast systems o We have produced over 150 recombinant proteins using our yeast expression systems. Our staff scientists are experienced in optimizing expression conditions. o In particular, the vector and P. pastoris strain pairs we have adopted allow high transformation efficiency and high cell density in culture. Proteins can be produced inside of the cells or in a secreted form. The secreting expression strategy has advantage in protein folding and protein purification.
o Of all the human genes that we have expressed in our yeast
systems, yields of 4~50mg/L have been regularly achieved. o Baculovirus-insect cell expression systems o Baculovirus-insect cell systems are considered a good system for recombinant glycoprotein production due to the eukaryotic nature of the host. o We have established a standard optimization procedure to determine the conditions for expressing soluble proteins using a traditional baculovirus system. The variables that we may alter to increase the efficiency and productivity of baculovirus expression are described below:
a. Plaque
purification of recombinant viruses; o The effect of these variables on successful protein expression is target dependent. Our staff scientists are experienced in high throughput operations for condition optimization. We normally achieve recombination efficiency of 100%, viral titer over 1x108 pfu/ml, expression success rate of 86%, and yield at 1~6mg/L. o We also employ a Bac-to-Bac Baculovirus Expression System to generate recombinant baculoviruses to produce recombinant proteins. This method is based on site-specific transposition of an expression cassette into a baculovirus shuttle vector (bacmid) propagated in E. coli. o The Bac-to-Bac Baculovirus Expression System provides the following advantages over the traditional method using homologous recombination:
a. Requires
less than 2 weeks to identify and purify a recombinant baculovirus as
compared to the 4-6 weeks required to generate a recombinant baculovirus using homologous
recombination; o Mammalian systems (engineered CHO, 293 cell lines) o We use engineered CHO and 293 cell lines as expression bioreactor. o We have integrated a gene, which represses the expression of apoptosis factors, into the engineered cells.
o To increase cell density in culture, we use a proprietary
adaptation procedure to suspend the originally adherent cells. o Cell-free systemso Eukaryotic cell-free system o Prokaryotic cell-free system | |||||||||
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