Expanded genetic code and Effect of synonymous mutations on expression of Fab fragments
Niemelä, Pyry (2019-12-13)
Expanded genetic code and Effect of synonymous mutations on expression of Fab fragments
Niemelä, Pyry
(13.12.2019)
Julkaisu on tekijänoikeussäännösten alainen. Teosta voi lukea ja tulostaa henkilökohtaista käyttöä varten. Käyttö kaupallisiin tarkoituksiin on kielletty.
suljettu
Julkaisun pysyvä osoite on:
https://urn.fi/URN:NBN:fi-fe202001314171
https://urn.fi/URN:NBN:fi-fe202001314171
Tiivistelmä
Antigen binding fragment (Fab) is the part of an antibody which contains
an antigen binding site. Fab has many advantages over a conventional
antibody such as the reduced size, lack of glycosylation and therefore, the
possibility of producing it in bacteria instead of complicated and expensive
mammalian cultures. Unfortunately, the yields of Fabs are relatively low in
bacterial cultures. Expression of human proteins in bacteria is hindered by
different preferences of synonymous codon usage between the two
different organisms. This phenomenon is present in all branches of life and
is known as codon bias.
We created a synonymous mutations library in a CDRH3 region of a
lysozyme binding Fab and fused the Fab to an ampicillin cleaving βlactamase. The β-lactamase allows a selection of better expressing
variants against an increasing concentration of ampicillin. An increase in
the frequency of hits was observed in correlation with increasing ampicillin.
At higher concentrations a decreasing trend of Fab expression was
observed. At a concentration of 800 µg/ml, the chance of finding a hit more
than doubled in comparison to a library without ampicillin selection.
The β-lactamase selection of improved Fab expression is a novel method
for the selection of synonymous mutation libraries. With optimization of the
selection conditions, the results with this basic principle could be improved
further.
This work consists two parts: a literary review of expanded genetic code
and the experimental part concerning β-lactamase selection of Fab
fragments from a synonymous mutation library.
an antigen binding site. Fab has many advantages over a conventional
antibody such as the reduced size, lack of glycosylation and therefore, the
possibility of producing it in bacteria instead of complicated and expensive
mammalian cultures. Unfortunately, the yields of Fabs are relatively low in
bacterial cultures. Expression of human proteins in bacteria is hindered by
different preferences of synonymous codon usage between the two
different organisms. This phenomenon is present in all branches of life and
is known as codon bias.
We created a synonymous mutations library in a CDRH3 region of a
lysozyme binding Fab and fused the Fab to an ampicillin cleaving βlactamase. The β-lactamase allows a selection of better expressing
variants against an increasing concentration of ampicillin. An increase in
the frequency of hits was observed in correlation with increasing ampicillin.
At higher concentrations a decreasing trend of Fab expression was
observed. At a concentration of 800 µg/ml, the chance of finding a hit more
than doubled in comparison to a library without ampicillin selection.
The β-lactamase selection of improved Fab expression is a novel method
for the selection of synonymous mutation libraries. With optimization of the
selection conditions, the results with this basic principle could be improved
further.
This work consists two parts: a literary review of expanded genetic code
and the experimental part concerning β-lactamase selection of Fab
fragments from a synonymous mutation library.