Author/Authors :
Abri Aghdam, Marjan Department of Biological Science - Faculty of Basic Science - Higher Education Institute of Rab-Rashid, Tabriz, Iran , Tohidkia, Mohammad Reza Research Center for Pharmaceutical Nanotechnology - Tabriz University of Medical Sciences, Tabriz, Iran , Ghamghami, Elham Department of Biological Science - Faculty of Basic Science - Higher Education Institute of Rab-Rashid, Tabriz, Iran , Ahmadikhah, Asadollah Faculty of Life Sciences and Biotechnology - Shahid Beheshti University, Tehran, Iran , Khanmahamadi, Morteza Chemical Engineering Faculty - Sahand University of Technology, Tabriz, Iran , Baradaran, Behzad Immunology Research Center - Tabriz University of Medical Sciences, Tabriz, Iran , Mokhtarzadeh, Ahad Chemical Engineering Faculty - Sahand University of Technology, Tabriz, Iran
Abstract :
Purpose: Production of functional recombinant antibody fragments in the periplasm of E. coli is
a prerequisite step to achieve sufficient reagent for preclinical studies. Thus, the cost-effective
and lab-scale production of antibody fragments demands the optimization of culture conditions.
Methods: The culture conditions such as temperature, optical density (OD600) at induction,
induction time, and IPTG concentration were investigated to optimize the functional expression
of a phage-derived scFv molecule using a design of experiment (DoE). Additionally, the effects
of different culture media and osmolyte supplements on the expression yield of scFv were
examined.
Results: The developed 2FI regression model indicated the significant linear effect of the
incubation temperature, the induction time, and the induction OD600 on the expression yield
of functional scFv. Besides, the statistical analysis indicated that two significant interactions of
the temperature/induction time and the temperature/induction OD600 significantly interplay to
increase the yield. Further optimization showed that the expression level of functional scFv
was the most optimal when the cultivation was undertaken either in the TB medium or in the
presence of media supplements of 0.5 M sorbitol or 100 mM glycine betaine.
Conclusion: In the present study, for the first time, we successfully implemented DoE to
comprehensively optimize the culture conditions for the expression of scFv molecules in a
phage antibody display setting, where scFv molecules can be isolated from a tailor-made phage
antibody library known as “Human Single Fold scFv Library I.”
Keywords :
Single-chain variable fragment (scFv) , Phage display , Response surface methodology , Periplasmic expression , Optimization , D-optimal design
