Bacterial fermentation of recombinant major wasp allergen Antigen 5 using oxygen limiting growth conditions improves yield and quality of inclusion bodies.
A process for bacterial expression and purification of the recombinant major wasp allergen Antigen 5 (Ves v 5) was developed to produce protein for diagnostic and therapeutic applications for type 1 allergic diseases. Special attention was focused on medium selection, fermentation conditions, and efficient refolding procedures. A soy based medium was used for fermentation to avoid peptone from animal origin. Animal-derived peptone required the use of isopropyl-beta-D-thiogalactopyranoside (IPTG) for the induction of expression. In the case of soy peptone, a constitutive expression was observed, suggesting the presence of a component that mimics IPTG. Batch cultivation at reduced stirrer speed caused a reduced biomass due to oxygen limitation. However, subsequent purification and processing of inclusion bodies yielded significantly higher amount of product. Furthermore, the protein composition of the inclusion bodies differed. Inclusion bodies were denatured and subjected to diafiltration. Detailed monitoring of diafiltration enabled the determination of the transition point. Final purification was conducted using cation-exchange and size-exclusion chromatography. Purified recombinant Ves v 5 was analyzed by RP-HPLC, CD-spectroscopy, SDS-PAGE, and quantification ELISA. Up to 15 mg highly purified Ves v 5 per litre bioreactor volume were obtained, with endotoxin concentrations less than 20 EU mg(-1) protein and high comparability to the natural counterpart. Analytical results confirm the suitability of the recombinant protein for diagnostic and clinical applications. The results clearly demonstrate that not only biomass, but especially growth conditions play a key role in the production of recombinant Ves v 5. This has an influence on inclusion body formation, which in turn influences the renaturation rate and absolute product yield. This might also be true for other recombinant proteins that accumulate as inclusion bodies in Escherichia coli.
Allergopharma Joachim Ganzer KG, Reinbek, Germany.
Stefanie
S
Kischnick
Bernhard
B
Weber
Petra
P
Verdino
Walter
W
Keller
Ernst A
EA
Sanders
F Birger
FB
Anspach
Helmut
H
Fiebig
Oliver
O
Cromwell
Roland
R
Suck
D000485:Allergens / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D018533:Biomass; D019149:Bioreactors / Q000382:microbiology; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development*; D002479:Inclusion Bodies / Q000737:chemistry / Q000378:metabolism; D019476:Insect Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D010100:Oxygen / Q000378:metabolism; D010101:Oxygen Consumption / Q000502:physiology; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D014862:Wasp Venoms / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics
D000485:Allergens; D019476:Insect Proteins; D011994:Recombinant Proteins; D014862:Wasp Venoms; C040583:white-faced hornet venom antigen 5; D010100:Oxygen