1. Production of scFabuv in Pichia pastoris
Introduction
The objective of this project is to produce the single chain
antibody fragment (scFabuv) that binds specifically to the green
fluorescent protein (GFPuv). In the scFabuv form, the heavy chain
and light chain are joined with a linker. This assures the correct
binding of both chains after protein expression.
The expression host is the yeast Pichia Pastoris which can secrete
the protein into the media, can yield high cell density and create
disulfide bonds that necessary for the correct conformation of the
scFabuv.
Results
Different PCR products were revealed on agar gels with ethidium
bromide.
Proteins from the supernatants were loaded on SDS-PAGE and
transferred to nitrocellulose membrane and specific scFabuv was
detected with protein H+L conjugated to HRP
Conclusion
Successfully generated P. pastoris for the
production of scFabuv that is secreted in the
production media. Future work will focus on
activity (binding to GFPuv) of the scFabuv by
using western blot or ELISA.
Method
• Amplification of Light and Heavy chain individually with PCR.
• PCR products purified and linked together with PCR
• LC + HC + LINKER fusion cut with restriction enzymes: EcoRI
and XbaI
• Cut plasmid pPICZalphaA with restriction enzymes: EcoRI and
XbaI
• Ligation of pPICZalphaA and HC + LC + LINKER fusion
• Transformation into E. coli
• Selection of colonies on LB petri plates with zeocine 25 µg/ml
• Positive clones screened with PCR
• Positive clone #6 cut with restriction enzyme SacI for
linearization and transformation into P. pastoris
• Positive clones selected on YPD with 500 µg/ml zeocine and
screened with PCR
• Grew four positive clones in 50ml of YPD medium and induced
with methanol .05% each day (4 days)
• Ran western blot on supernatants before and after induction
Roberta Cassar & Dr. Driss Elhanafi
Amplification of LC & HC
individually
PCR linking LC & HC +
linker
700 bp
HCLC
LC + HC +
LINKER
1500 bp
1 2 3 4 5 6-------------------13
#6 clone
positive
match
PCR for positive clone
on selection plates
Cut #6 with SAC1 for
easy transformation into
P. pastoris
5000 bp
pPICZalphaA
pPICZalphaA with HC + LC +
LINKER
PCR with F11 and
R11 on zeocine
500 resistant
clones
1 2 3 4
Western Blot using protein H+L-HRP
Clones
1 2 3 4 5 6 7 8
1: Clone #1 + CH3OH
2: Clone #2 + CH3OH
3: Clone #3 + CH3OH
4: Clone #4 + CH3OH
5: Clone #1 - CH3OH
6: Clone #2 - CH3OH
7: Clone #3 - CH3OH
8: Clone #4 - CH3OH
Acknowledgments
Would like to thank Dr. Hentz for his guidance
and support during the summer internship.
Thank you Nate Cota for your help during the
western blot process. Finally would like to give a
huge thank you to my mentor Dr. Driss for
instructing me and giving me the opportunity to
learn and develop skills that are helpful in the
work force. I enjoyed this work experience and
I’m honored to have been apart of it.