Establishment And Optimization Of A Transient,Serum-free Expression System For HEK293-6E And Its Application For The Expression Of Noggin,a Growth Factor For Organoid Culture

Objective:Serum-free medium is increasingly used in the basic research field of biomedicine,and for the production of biopharms and vaccines.HEK293-6E is currently human-derived cells capable of expressing high-quality recombinant proteins,but its serum-free medium is expensive,and its efficacy for recombinant protein production also has room for further improvement.This study aims to optimize the self-developed HEK293-6E serum-free medium and establish a low-cost,high-efficiency HEK293-6E transient expression system,in order to express a variety of high-quality recombinant proteins including growth factors required for organoid culture.Methods:(1)Cell culture medium component replacement and cell culture domestication were applied to adapt HEK293-6E to the serum-free culture medium established in this study.Rice-derived recombinant human serum albumin(HSA)was used to replace animal-derived bovine serum albumin(BSA)to achieve animal-free culture media.Polyamine chemical putrescine was further utilized to abtain protein-free and chemically-defined serum-free culture media for HEK-293-6E.A fed-batch culture scheme was established to prolong cell growth time;(2)CCK-8,trypan blue staining,and cell morphology observation under optical microscope were applied to evaluate the culture status of HEK293-6E;(3)p TT5/GFP expression plasmid was constructed by molecular cloning techniques and this construct was used as a,reporter to evaluate the effects of different transfection reagents and transfection schemes on the expression levels of recombinant GFP,which is analyzed by Western blotting so as to establish an optimal transient transfection scheme for HEK293-6E and to evaluate the efficacy of this system for the expression of intracellular recombinant protein expression;(4)The DNA coding sequence of the signal peptide of growth factor Noggin was replaced with that of IFN?-2,Ig G heavy chain or Gaussia luciferase,and a 6histidine tag was also in-frame added at the carboxyl termini of Noggin constrtucts for expression analysis and purification.Plasmids were transfected into HEK293-6E seperately,and the effects of different signal peptides on Noggin secretive expression wwere analyzed by Western blotting;(5)p TT5/Noggin plasmid with Guassia luciferase signal peptide(the most efficient signal peptide for Noggin seretion)was transiently transfected into HEK293-6E cells.High cell density and fed-batch culture was resorted for the expression of recombinant Noggin protein in1 L culture volume.Noggin was purified by metal nickel affinity chromatography,The biological activity of recombinant Noggin was evaluated by organoid culture of mouse small intestinal crypt stem cel s.Results:(1)HEK293-6E gradually adapted to our self-developed serum-free medium.Cell viability,proliferation rate,and cell morphology cultured with this medium were not significantly different from those with commercial medium;100 mg/L HSA could effectively replace BSA in the medium,and putrescine could also effectively support cell growth.(2)A fed-batch culture protocol was established: o n the third day of cell culture,supplementation of 0.2923 g/L glutamine,1.74 g/L EX-Cell protein hydrolysate,10% 5×serum-free medium couldn effectively extend the cell growth time from 3 days to 8 days.(3)The p TT5/GFP exxpression plasmid was successfully constructed.The transfection conditions were optimal when PEI was used as the transfection reagent and the ratio of transfection plasmid to PEI was 1/2.In this system,the intracellular expression of recombinant GFP reached 47.85 mg/L.(4)Four p TT5/Noggin plasmids with IFN?-2,Ig G heavy chain,Gaussia luciferase or Noggin natural signal peptide and 6-histidine tag at the carboxyl end were successfully constructed.In our HEK293-6E transient expression system,Gaussia signal peptide displayed the strongest secretion for recombinant Noggin expression.(5)The HEK293-6E transient expression system was used to express the recombinant protein Noggin.Noggin protein was successfully purified with a yield of 6.9 mg/L and the protein is biological active as it could support organoid culture ofmouse small intestinal crypt stem cel s as compared with commercial Noggin.Conclusion:This project successfully developed a serum-free medium formula for HEK293-6E culture.Evaluated by cell viability,proliferation rate and cell morphology,the formula was found similar to that of commercial serum-free medium,which greatly reduce the cost of HEK293-6E serum-free culture.The use of EX-Cell protein hydrolysate,ferric citrate and putrescine successfully replaced the corresponding growth factors,transferrin and BSA in the original medium,laying the foundation for the establishment of a serum-free medium without protein or animal protein.Transient transfection scheme and fed-batch culture protocol were successfully established for HEK293-6E.The expression yield of intracellular protein GFP under this system reached tens of milligrams per liter,and secretive expression of Noggin with a Gaussia signal peptide reached 6.9 mg/L.The recombinant Noggin growth factor was able to induce the organoid growth of mouse small intestinal crypt stem cells,indicating that Noggin protein was biological active.