To design a protein which can bind all 4 RSPOs extracellularly.

Choose the extracellular domains of two membrane proteins (LGR4 and ZNRF3) that have been reported to interact with RSPO and connect them with a linker with optimized length.

The binding capability with RSPO: (Linker1-4 represent different linker lengths)

Linker3 is the optimal linker. The secretion efficiency should be optimized next.

To improve the secretion capability

Choose several signaling peptides and compare the secretion efficiency

The secretion capability of RTAC with different signaling peptides:

FZD5 signaling peptide is the best. The protein stability should be improved next.

To improve the stability by fusing additional domain(s).

Adding human IgGH3 Fc region to the carboxyl terminus of RTAC.

The stability of RTAC was improved.

RTAC is most stable protein. The engineering success of RTAC achieved.
See more in part “BBa_K4220000”

Design:

1. Idea: Reprogramming the mating pathway to enable the yeast to target tumor by specific signals in tumor microenvironment (TME).

2.Literature search:

Signaling molecules selection:
Choose extracellular ATP (eATP) as the signal because of its specific presence at TME compared with in healthy tissues.

Receptor determination:
Trigger mating pathway with eATP by replacing the original receptor (ste2) with modified human P2Y2 receptor which specifically responses to eATP.

Signal transduction:
Introducing the chimeric Gα protein with 5 C-terminal amino acids of mammalian Gαi3, which is capable to be recruited by human P2Y2 receptor and transduce the signal downstream.

Signal amplifying:
Removing sst2 can intensify the signal output of mating pathway

Tool:
P2A peptide can connect and then generate two different proteins when it is cleaved by specific enzyme.

· Plan1:

Gene editing:

Removing original receptor gene ste2.

Removing sst2 to intensify the signal output.

· Test1:

The gene editing success was verified by PCR.

· Plan2:

Transforming P2Y2-P2A-mCherry

· Test2:

The P2A peptide can work effectively:

· Plan3:

Transforming P2Y2-P2A-chimeric Gα

· Test3:

Engineering completed:

The original mating pathway was reprogrammed. P2Y2 receptor and chimeric Gα can be successfully introduced into the yeast and the P2A functions well to separate these two proteins.

See more in part “BBa_K4220008” and part “BBa_K4220007”

Design:

1. Idea: engineer RTAC downstream of the mating pathway

2. Literature search:

MFα-1 is the signaling peptide of α-factor which is particular for yeast expression

FUS1 is the final outputting product of mating pathway

· Plan1:

Transform P2Y2-P2A-Gα into the yeast.

Proving the function of P2A and demonstrate the successful reprogramming of eATP-P2Y2-Gα-MAPK: transform the pFUS1-mCherry into the yeast.

· Test1:

The reprogrammed mating pathway can effectively sense the eATP and express mCherry indicated by red fluorescence:

· Plan2:

Changing of signaling peptide of RTAC: change the Frizzled5 signaling peptide into MFα1

Including RTAC into the mating pathway: transform pFUS1-RTAC into the yeast.

· Test2:

RTAC can be successfully produced and secreted in an eATP dose-dependent manner: