Team:Montpellier/WetLab ASA

Antibodies

Designs

The following figure (Figure 1) is the caption for the next 3 figures of our designs. Orange frames are the variable parts between the constructs.

Figure 1 : Meaning of the icons for the 3 following figures.

We decided to design several constructions to produce YLP20, our favorite candidate with four differents signal peptides : CbsA, Epr, YncM and Yjfa (Figure 2). We chose RpsU promoter [1] that is a strong promoter specific to L. jensenii.
We tested different signal peptides that we introduced you before without forgetting the amino sequence “APVT” to avoid truncating the protein. Finally, we added the sequence that encodes YLP20 scFv and E-tag.

Figure 2 : Constructs that produce and secrete YLP20 with 4 different signal peptides.

Figure 3 : Constructs that produce and secrete LaM-4 (a control) with 4 different signal peptides.

To check the scFv production and conformation we designed constructions with LaM-4 instead of YLP20 sequence (Figure 3).
Indeed LaM-4 is a control [2]. It is an antibody against RFP & mcherry. This control allow us to check the efficiency of the promoter and the signal peptide.
The construction is very similar that the one with YLP20. The difference is just that we replaced the YLP20 sequence with the LaM-4 sequence.

Moreover, we would like to check signal peptides that we chose. Indeed, it is really important to verify the efficiency of the SP and determine new SP that are working in L. jensenii. We decided to fusion the SP to RFP to display the efficiency of the SP (Figure 4). For that, we used a plate reader and a flow cytometer.

Figure 4 : Constructs that produce and secrete RFP with 4 different signal peptides.

Experimental design to assess Antisperm Antibodies production and secretion

To verify if our constructs are working correctly (i.e production and secretion in the supernatant) we designed several experiments (Figure 5).

The first experiment consists of a Western-Blot (WB) to verify that the antibody is produced by the bacteria. Our scFv is fused to an E-tag, allowing for straigthforward detection using a commercial antibody.

The second experiment consists of an ELISA test using YLP12 (biotinylated as follow : YLPVGGLRRIGG-Lys(Biotin)) which is the target antigen for the YLP20 scFv. The idea is to use streptavidin plates to fix YLP12, so that he repulsion between biotin and streptavidin forces the peptides not to lie on the bottom of the plate and facilitate the binding antigen-antibody. Different ELISA tests would need to be performed : First, using a supernatant from a bacterial culture to see if YLP20 is produced and secreted. In parallel, an ELISA using the cell lysate would be useful to see if YLP20 is produced but not secreted.

Figure 5 : Experimental design to assess production and secretion of antisperm scFvs in *Lactobacillus jensenii*. All the constructions are cloned by Gibson Assembly in *E. coli*. After the constructions are sequence verified, *L. jensenii* is transformed by electroporation. To verify if both production and secretion of scFv are effective, a Western Blot would be performed. After that, an ELISA test run to see if the produced scFv ca link to the specific peptide YLP12.

Expected results

Figure 6 describes the possible outcomes of the Western Blot experiment. If everything in the YLP20 sequence is working, we will detect the scFv in both supernatant and pellet (cell lysate). If only the signal peptide is not working, we will have a signal only in the cell lysate. Finally, if the scFv is not well expressed, (e.g. because of the promoter, the RBS, or aggregation) we will have detect no signal neither from the supernatant nor from the pellet. As a positive control, we must run a WB on a strain containing a protein fused to an E-tag that we know is well expressed.

Results

All the constructions were cloned in E. coli DH5ɑ. Currently, we did not succeed to transform L. jensenii with different antibody constructions.

Cloning

Part		Designed	Cloned	Characterized
Antibody	Signal Peptide	Designed	Cloned	Characterized
YLP20	CbsA	YES	YES	YES
	YncM	YES	YES	YES
	YjFA	YES	NO	NO
	EPR	YES	NO	NO
LaM-4	CbsA	YES	YES	YES
	YncM	YES	YES	YES
	YjFA	YES	NO	NO
	EPR	YES	NO	NO
RFP	CbsA	YES	YES	NO
	YncM	YES	YES	NO
	YjFA	YES	NO	NO
	EPR	YES	NO	NO
LaG-16–G4S–LaG-2	CbsA	YES	NO	NO
	YncM	YES	NO	NO
	YjFA	YES	NO	NO
	EPR	YES	NO	NO

Western Blot

Since we did not succeed to transform Lactobacillus jensenii properly, we studied the production and secretion of our antibodies in E. coli with the plasmid psb1C3 (because it is a high copy number plasmid). Negative control is E. coli without plasmid.

The expected antibody sizes are the following:

LaM-4 (with the signal peptide YncM) : 570 nt | 20,9 kDa
LaM-4 (with the signal peptide CbsA) : 527 nt | 19,25 kDa
LaM-4 (without the signal peptide) : 432 nt | 15,84 kDa

YLP20 (with the signal peptide YncM) : 855 nt | 31,3 kDa
YLP20 (with the signal peptide CbsA): 813 nt | 29,81 kDa
YLP20 (without the signal peptide) : 717 nt | 26,29 kDa

We can only see the presence of a protein with E-tag in the pellet, and a slight presence in the supernatant. So, we cannot said that there is a secretion but a least there is a production inside the cell.

Moreover, the sizes observed do not correspond to our expectations, mainly for YLP20 which seems to small. It could be supposed that the protein has been cleaved. Currently, we can just confirm that our promoter works in E. coli.

References
[1]	Xiaowen Liu, Laurel A. Lagenaur, David A. Simpson, Kirsten P. Essenmacher, Courtney L. Frazier-Parker, Yang Liu, Daniel Tsai, Srinivas S. Rao, Dean H. Hamer, Thomas P. Parks, Peter P. Lee and Qiang Xu. (2006). Engineered Vaginal Lactobacillus Strain for Mucosal Delivery of the Human Immunodeficiency Virus Inhibitor Cyanovirin-N. Antimicrob Agents Chemother Vol. 50, No. 10, p. 3250–3259
[2]	Fridy, P. C., Li, Y., Keegan, S., Thompson, M. K., Nudelman, I., Scheid, J. F., ... & Rout, M. P. (2014). A robust pipeline for rapid production of versatile nanobody repertoires. Nature methods, 11(12), 1253.