Difference between revisions of "Part:BBa K4195038"

Latest revision as of 12:50, 12 October 2022

Biology

rLvAPN1

LvAPN1, a protein from the aminopeptidase N family, was identified in Litopenaeus vannamei hemocytes as a receptor for VP_AHPND toxin PirA and PirB. It is constitutively expressed in the stomach, hepatopancreas, and hemocytes of healthy shrimp, while the increased expression of which was found after the shrimp were challenged with the VP_AHPND toxin. Silencing of LvAPN1 prevents the VP_AHPND toxin from passing through the cell membrane (1).

rLvAPN1 is a truncated form of LvAPN1 (residues 205-591) that composes of a crystal insecticidal (Cry) toxin binding region and the active site of peptidase-M1 domain, which was reported to directly bind to both PirA and PirB toxins. What’s more, there is no glycosylation sites in rLvAPN1, which makes it easier to obtain the purified protein by using prokaryotic expression system (such as E. coli).

Usage and design

In order to purify rLvAPN1, a His-tag (6×His) was added to the C-terminal of rLvAPN1. We used arabinose-inducible system to express the recombinant rLvAPN1-his then constructed composite part BBa_K4195134.

Characterization

Identification

After the first time of purification, we found that the protein was poorly expressed and mostly formed inclusion body, so we did not obtain the target protein. Therefore, we cloned this part into the expression vector pET-28a(+) via Gibson assembly, then transformed the correct plasmid into E. coli BL21(DE3). When constructing this circuit, colony PCR and gene sequencing were used to verify that the transformatants were correct. Target bands (1386 bp) can be observed at the position between 1000 and 1500 bp (Fig. 1).

Fig. 1 DNA gel electrophoresis of the colony PCR products of BBa_K4195038_pET-28a(+).

SDS-PAGE

The plasmids verified by sequencing was successfully transformed into E. coli BL21(DE3). After being cultivated and induced by IPTG, GE AKTA Prime Plus FPLC System was employed to get purified protein from the lysate supernatant. Purified protein was verified by sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel electrophoresis (PAGE) and Coomassie blue staining. As shown in the gel image of rLvAPN1-his (Fig. 2), the bands of target protein (45.5 kDa) could be observed at the position between 35 and 50 kDa on the purified protein lanes (FR).

Fig. 2 SDS-PAGE analysis of protein in lysate of E. coli BL21(DE3) and the elution samples. Target bands (45.5 kDa) can be observed at the position between 35 and 50 kDa.

Western blot

This year, we shared deep collaborations with a first-year team, CUG-China. With the help of them for offering the operation of western blot, we could confirm that the rLvAPN1-his was expressed indeed (Fig. 3), after struggling to obtain the purified protein for several rounds mentioned above.

Fig. 3 Western blot analysis (anti-His-tag) of protein in lysate supernatant of E. coli BL21(DE3). Target bands (45.5 kDa) can be observed at the position between 43 and 55 kDa.

Reference

1. W. Luangtrakulet al., Cytotoxicity of Vibrio parahaemolyticus AHPND toxin on shrimp hemocytes, a newly identified target tissue, involves binding of toxin to aminopeptidase N1 receptor. PLoS Pathog. 17, e1009463 (2021).

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 847
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 711
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 760
1000
COMPATIBLE WITH RFC[1000]

@@ Line 4: / Line 4: @@
 r''Lv''APN1
-''LvAPN1'', a protein from the aminopeptidase N family, was identified in ''Litopenaeus vannamei'' hemocytes  as a receptor for VP<sub>AHPND</sub> toxin PirA and PirB. It is constitutively expressed in the stomach, hepatopancreas, and hemocytes of healthy shrimp, while the increased expression of which was found after the shrimp were challenged with the VP<sub>AHPND</sub> toxin. Silencing of ''Lv''APN1 prevents the VP<sub>AHPND</sub> toxin from passing through the cell membrane (''1'').
+''Lv''APN1, a protein from the aminopeptidase N family, was identified in ''Litopenaeus vannamei'' hemocytes  as a receptor for VP<sub>AHPND</sub> toxin PirA and PirB. It is constitutively expressed in the stomach, hepatopancreas, and hemocytes of healthy shrimp, while the increased expression of which was found after the shrimp were challenged with the VP<sub>AHPND</sub> toxin. Silencing of ''Lv''APN1 prevents the VP<sub>AHPND</sub> toxin from passing through the cell membrane (''1'').
 r''Lv''APN1 is a truncated form of ''Lv''APN1 (residues 205-591) that composes of a crystal insecticidal (Cry) toxin binding region and the active site of peptidase-M1 domain, which was reported to directly bind to both PirA and PirB toxins. What’s more, there is no glycosylation sites in r''Lv''APN1, which makes it easier to obtain the purified protein by using prokaryotic expression system (such as ''E. coli'').
 ===Usage and design===
 In order to purify r''Lv''APN1, a His-tag (6×His) was added to the C-terminal of r''Lv''APN1. We used arabinose-inducible system to express the recombinant r''Lv''APN1-his then constructed composite part <partinfo>BBa_K4195134</partinfo>.
@@ Line 13: / Line 14: @@
 After the first time of purification, we found that the protein was poorly expressed and mostly formed inclusion body, so we did not obtain the target protein. Therefore, we cloned this part into the expression vector pET-28a(+) ''via'' Gibson assembly, then transformed the correct plasmid into ''E. coli'' BL21(DE3). When constructing this circuit, colony PCR and gene sequencing were used to verify that the transformatants were correct. Target bands (1386 bp) can be observed at the position between 1000 and 1500 bp (Fig. 1).
-[[File:T--XMU-China-BBa K4195134 Fig1.png|200px]]
+[[File:RLvAPN1-pet.png|200px]]
-'''Fig. 1 DNA gel electrophoresis of the colony PCR products of <partinfo>BBa_K4195134</partinfo>_pET-28a(+).'''
+'''Fig. 1 DNA gel electrophoresis of the colony PCR products of BBa_K4195038_pET-28a(+).'''
 ====SDS-PAGE====
 The plasmids verified by sequencing was successfully transformed into ''E. coli'' BL21(DE3). After being cultivated and induced by IPTG, GE AKTA Prime Plus FPLC System was employed to get purified protein from the lysate supernatant. Purified protein was verified by sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel electrophoresis (PAGE) and Coomassie blue staining. As shown in the gel image of r''Lv''APN1-his (Fig. 2), the bands of target protein (45.5 kDa) could be observed at the position between 35 and 50 kDa on the purified protein lanes (FR).
+[[File:Nnew-rLvAPN1-is.png|300px]]
+'''Fig. 2 SDS-PAGE analysis of protein in lysate of ''E. coli'' BL21(DE3) and the elution samples.''' Target bands (45.5 kDa) can be observed at the position between 35 and 50 kDa.
+====Western blot====
+This year, we shared deep collaborations with a first-year team, CUG-China. With the help of them for offering the operation of western blot, we could confirm that the r''Lv''APN1-his was expressed indeed (Fig. 3), after struggling to obtain the purified protein for several rounds mentioned above.
+[[File:New-wb-rLvAPN1-HIS.png|300px]]
+'''Fig. 3 Western blot analysis (anti-His-tag) of protein in lysate supernatant of ''E. coli'' BL21(DE3).''' Target bands (45.5 kDa) can be observed at the position between 43 and 55 kDa.
+===Reference===
+.	W. Luangtrakul''et al.'', Cytotoxicity of ''Vibrio parahaemolyticus'' AHPND toxin on shrimp hemocytes, a newly identified target tissue, involves binding of toxin to aminopeptidase N1 receptor. ''PLoS Pathog.'' '''17''', e1009463 (2021).