iFLinkC: an iterative functional linker cloning strategy for the combinatorial assembly and recombination of linker peptides with functional domains

Abstract Recent years have witnessed increasing efforts to engineer artificial biological functions through recombination of modular-organized toolboxes of protein scaffolds and parts. A critical, yet frequently neglected aspect concerns the identity of peptide linkers or spacers connecting individual domains which remain poorly understood and challenging to assemble. Addressing these limitations, iFlinkC comprises a highly scalable DNA assembly process that facilitates the combinatorial recombination of functional domains with linkers of varying length and flexibility, thereby overcoming challenges with high GC-content and the repeat nature of linker elements. The capacity of iFLinkC is demonstrated in the construction of synthetic protease switches featuring PDZ-FN3-based affinity clamps and single-chain FKBP12-FRB receptors as allosteric inputs. Library screening experiments demonstrate that linker space is highly plastic as the induction of allosterically regulated protease switches can vary from >150-fold switch-ON to >13-fold switch-OFF solely depending on the identity of the connecting linkers and relative orientation of functional domains. In addition, Pro-rich linkers yield the most potent switches contradicting the conventional use of flexible Gly-Ser linkers. Given the ease and efficiency how functional domains can be readily recombined with any type of linker, iFLinkC is anticipated to be widely applicable to the assembly of any type of fusion protein.


Restriction Digest DNA Fragments with BtsI and BsrDI
First of all, devise an assembly process deciding on the order of functional domains and linker elements in the fusion protein (see Fig. 2 in the main manuscript). Functional domains are generally stored in pFD while linker elements are stored in pL2. Please note, pFD and pL2 are functionally equivalent enabling the direct fusion of any two functional domains or linkers.
Once an assembly process has been devised, set up restriction digests using a combination of BtsI, BsrDI and either SpeI or EcoRI: The N-terminal DNA fragment is always digested with BsrDI while the C-terminal DNA fragment is always digested with BtsI. Depending on the size of the anticipated C-terminal DNA fragment either EcoRI or SpeI can be employed. For shorter DNA fragments, we recommend EcoRI while for longer DNA fragments >400 bp that are amenable to gel purification by themselves, we recommend SpeI.
To prevent re-ligation of the entry plasmids in the subsequent ligation step, we recommend dephosphorylating one of the two restriction digests with recombinant shrimp alkaline phosphatase (rSAP). Instead of treatment with rSAP, plasmids may also be restriction digested with BbsI and/or BsaI to prevent re-ligation of the entry plasmid.
Restriction digest with BsrDI (in a total volume of 50 µL; exemplified with pL2): -Add equimolar amounts of purified pL2 coding for different linker elements. The total DNA mass should not exceed 1.2 µg. -Fill up to a volume of 43 µL with MQ water.

Purification of DNA Fragments by Agarose Gel Electrophoresis
-Stop restriction digest by adding 10 µL DNA loading dye (6x stock) to 50 µL restriction digest. -Separate DNA fragments by means of agarose gel electrophoresis (for approx. 40 min at 120 V on 1% agarose in 1x TAE buffer). Depending on the size, DNA fragments may also be separated for a longer period of time and reduced voltage. -Excise the desired DNA fragments using a clean scalpel.
-Extract DNA fragments from agarose gel, preferably with a commercial gel extraction kit such as NucleoSpin (Macherey-Nagel).

iFLinkC Assembly Reaction: Ligation of pFD and pL2 Fragments
-Add equimolar amounts of gel purified pFD and pL2. Total amount of DNA should be between 5 ng and 100 ng.

Transforming iFLinkC Assembly Reaction
-Transform 2-3 µL of the iFLinkC assembly reaction into 50 µL aliquots of transformationcompetent E. coli. Transformation can either be realised by means of heat-shock or electroporation. -Perform outgrowth in 1 mL SOC medium for 1 h at 37 ˚C.
-To assess the efficiency of transformation, plate 40 µL on LB agar plate supplemented with 50 µg/mL kanamycin. The fidelity of the assembly reaction may also be confirmed by sequencing of individual clones. -Use the remaining 960 µL to inoculate 10 mL LB supplemented with 50 µg/mL kanamycin and overnight incubation at 37 ˚C.

Cloning
Step Part Description   Table  3.  Table  3.  Table 4.  Table 4.  Table 4.  Table 4. Expression tests were conducted in LB while protein expression was induced with 1 mM IPTG during exponential growth phase and left to express for 3 h. Aliquots of the cell suspension were denatured for 10 min at 95 ˚C in SDS-PAGE loading buffer. Expression analysis shows AI-FKBP12-FRB-TVMV express well with an approx. molecular weight of >90 kDa for the full length MBP-CS TEV -StrepTag-II-AI-FKBP12-FRB-TVMV fusion protein (see red arrow). Two additional bands are visible at approx. 40 kDa and 52 kDa corresponding to non-specific cleavage products of the MBP and AI-FKBP12-FRB-TVMV protease switch. iFLinkC-XE with an mNeonGreen-CfaN insert of approx. 35 kDa (see grey arrow) served as the negative control.