Translation Engineering
Applications
Success Stories
Publications
Success Stories
 
Improved solubility of IRAK-4
CODA was able to increase total solubility by 38.4%.  A deletion library was constructed by multiplexing PCR against the IRAK-4 Hot Rod template with 5 and 3’ over-lapping primers for sequential N- and C-terminus truncations.  The deletion library was then screened by a proprietary selection filter. Potential soluble clones were identified, sequenced and verified for soluble expression.

 

Figure: Improved solubility for IRAK4 deletion variants. Proteins were expressed from a pET vector in E. coli strain BL21(DE3), inducing with 1 mM IPTG for 3 hours at 30°C.  Lane M: molecular weight markers (in kDa). Lanes 1-4, full-length IRAK4: uninduced total protein, induced total protein, induced insoluble fraction, and induced soluble fraction, respectively.  Lanes 5-8, IRAK4 deletion clone #21: uninduced total protien, induced total protein, induced insoluble fraction, and induced soluble fraction, respectively.  Lanes 9-12, IRAK4 deletion clone #12: uninduced total protein, induced total protein, induced insoluble fraction, and induced soluble fraction, respectively.  Left panel, Coomassie Blue staining; Right panel, western blot analysis using Anti-6xHis antibody.

SolSelect DL Clone
Size (amino acids)
% Total Solubility
% Solubility Improvement
#1
279
18.2
139.5
#11
260
28.8
278.9
#12
266
38.4
405.3
#13
291
14.1
85.5
#21
269
8.5
11.8
#41
282
37.0
386.8
#45
259
42.2
455.3

 
 

Improved solubility of (TEL)-SAM clones using  Combinatorial Library (CL) approach
CODA was able to increase total solubility by 88.5%.  Using a Combinatorial Library approach, CODA constructed a combinatorial library, with minimal amino acid changes and screened the clones through a proprietary selection filter.  Potential soluble clones were identified, sequenced and verified for soluble expression. 

   

Figure:  Improved solubility for CL (TEL)-SAM clone TSM#40 discretely expressed as pET constructs in E. coli. Lane M: molecular size markers (in kDa); Lanes 1-4: Un-induced (TEL)-SAM Hot Rod clone, induced with 1 mM IPTG, induced insoluble fraction, and induced soluble fraction at 30°C for 3 hrs; Lanes 5-8: Un-induced CL clone TSM#40, induced with 1 mM IPTG, induced insoluble fraction, and induced soluble fraction at 30°C for 3 hrs.

 

 
Lane #
Sample
M
MW Marker
1
HRTS Total Protein, Un-induced
2
HRTS Total Protein, Induced
3
HRTS Total Protein, Induced Soluble
4
HRTS Total Protein, Induced Insoluble
5
TSM-40 Total Protein, Un-induced
6
TSM-40 Total Protein, Induced
7
TSM-40 Total Protein, Induced Soluble
8
TSM-40 Total Protein, Induced Insoluble

 
Improved solubility of a Interleukin-2 clone using a Combinatorial Library (CL) approach
CODA was able to increase solubility by 13%.
   

SolSelect™ CL Clone
Solubility (%)
Total
Improvement
IL-2 Hot-Rod™
5.3
 NA
IL-2M#9
11.7
 120.8
IL-2M#15
11.6
 118.9
IL-2M#26
7.4
 39.6
IL-2M#42
13.0
 145.3
IL-2M#43
9.7
 83.0

 

Figure: Improved solubility for IL-2 (134 amino acids without the signal peptide) mutant clones discretely expressed as pET constructs in E. coli. Lane M: molecular size markers (in kDa); Lanes 1-4: Un-induced IL-2 Hot Rod clone, induced with 1 mM IPTG, induced insoluble fraction, and induced soluble fraction at 30°C for 3 hrs; Lanes 5-8: Un-induced CL mutant clone #9, induced with 1 mM IPTG, induced insoluble fraction, and induced soluble fraction at 30°C for 3 hrs; Lanes 9-12: Un-induced SolSelect CL mutant clone #42, induced with 1 mM IPTG, induced insoluble fraction, and induced soluble fraction at 30°C for 3 hrs.

   

Client Therapeutic Protein Expression Yield Increased by 65X.
A human derived biotherapeutic protein was poorly expressed in
E. coli. The CODA Translation Engineered version of both the human gene and the homologous rat gene were greatly improved as can be seen in the Coomassie stained gel showing total cell protein.

 
A collaborator in CODA's Academic Access program designed three entirely synthetic proteins consisting of peptides concatenated from multiple protein targets.
As a result of CODA's Translation Engineering, all three proteins were expressed at high levels without further optimization.