Expression Of Recombinant Limulus Factor C And Endotoxin Detection

Bacterial endotoxin is a toxin released after cell wall lysis of gram negative bacteria,also known as"pyrogen".Its main component is lipopolysaccharide（LPS）,which after entering the human body can lead to fever,shock and even death.Because of its ubiquitous presence in the environment and difficult inactivation,highly sensitive endotoxin detection methods are required for quality control when developing biopharmaceutical products.The limulus assay,which is derived from Limulus hemolymph,has been widely applied for endotoxin detection since the early 1980s.With the deteriorating marine environment and serpiginous human predation in recent years,Limulus resources have dramatically decreased,greatly limiting the source of Limulus reagents.So the man developed a new generation of endotoxin detection kit,the recombinant fluorometric kit,which does not rely on Limulus.Endotoxin specifically activates Limulus C factor to cleave a fluorogenic substrate,thereby quantitatively detecting endotoxin.However,the recombinant Limulus C factor is structurally complex,controlling endotoxin in the expression purification process is difficult and costly.This has led to the current price of commercially available recombinant fluorometric kits being expensive.Therefore,it is essential to explore the possibility of constructing an efficient recombinant factor C（rFc）expression system.（1）In this study,the full gene sequence of factor C in the Tachypleus tridentatus was cloned and characterized.First,the recombinant plasmids were electro transferred into Corynebacterium glutamicum（C.glutamicum）,Komagataella phaffii（K.phaffii）by genetic engineering.And at the end of fermentation,no rFc protein inside and outside the cell was detected by Western Blot（WB）.Second,a baculovirus plasmid was constructed,which was transfected into insect cells using lipofectamine.And at the end of the fermentation,WB detected that the rFc was intracellularly soluble and of the size expected,however the expressed rFc was in the single chain form,not expected.（2）Then,the recombinant plasmid was reconstituted,which was recombinantly expressed utilizing liposomal transfection into the human embryonic kidney cell Expi293F.WB detected rFc expressed in extracellular soluble secretion,with a size consistent with the prediction,and in a double stranded form.The expression was 19.81 mg·L^-1,which was 113%higher than the highest expression reported currently,with biological activity of binding to endotoxin and cleaving a fluorescent substrate.However,a large proportion of rFc was prematurely activated due to an excess of endotoxin introduced during expression and purification.It was expressed as the difference in relative fluorescence did not have a linear relationship with the exogenously applied endotoxin content.（3）Finally,the spilt intein Cfa DnaE was used to break the endotoxin binding domain Sushi 1,Sushi 3 of the rFc to fuse the N-terminal sequence（S1N,S3N）of the rFc with the intein（Cfaⁿ）,and the C-terminal sequence（S1C,S3C）of the rFc with the C-terminal sequence（Cfa^c）of the intein,respectively.S1N-Cfaⁿ、Cfa^c-S1C、S3N-Cfaⁿ、Cfa^c-S3C fusion proteins were successfully expressed,in which S1N-Cfaⁿ and S3N-Cfaⁿ were intracellularly insoluble.After purification,refolding,and removal of endotoxin,S3N-Cfaⁿ and Cfa^c-S3C were self splice in vitro to form intact in rFc（the self spliced rFc）,And the highest self splicing efficiency was 78%when p H was 7.5 and temperature was 25°C.The rFc in this zymogen form exhibited a linear difference in relative fluorescence to that of endotoxin spiked with different concentrations of endotoxin,which has the ability to quantitatively detect endotoxin.This method provides a new idea for the subsequent development of rFc recombinant expression system and endotoxin detection kit.