ScFv expression in periplasmic space

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William31
William31's picture
ScFv expression in periplasmic space

Hi everybody,

I am expressing a scfv antibody (fusion protein) and since most scfv expressed are insoluble, i decided to use the plasmid pet 26b which has a pelb sequence for periplasmic translocation. The host is bl21 de3 plyss.

However, I couldnt use the his tag at the C terminal (to not interfer with a peptide fused to my scfv at the C ter), so I put the his tag at the N terminal.

=>
atg_pelb_his tag_scfv_peptide_stop

The his tag is after pelb sequence because i dont want it to be removed when the fp protein reaches periplamic space, pelb sequence is supposed to be removed. I knew that his-tag (n ter) could interfer with pelb sequence but some people have done this before. Actually they got extracellular secretion, because his-tag in N terminal enhance extracellular secretion when there is a pelb sequence at N-ter as well.

Unfortunately, my protein is expressing in inclusion bodies (sds page after extraction), i guess his-tag is interfering with pelb signal sequence. I would like to know if someone has done this, denaturing the inclusion bodies and then refolding. Have I a good chance to get the correct re folding ?

What can I do else ? What I have read about denaturing refolding is : time consuming, expensive etc.... Is there a easy and cheap protocol for refolding purification ?

Thanks very much !

protoldo
protoldo's picture
Bonjour ,yes denaturalization

Bonjour ,yes denaturalization/refolding is a complex process wich can go well for little and easy proteins,but increasing complexity the results are worse.I checked the novagen manual, because the periplasmic signals drive your proteins to the periplasmic space and I don´t know if export to the medium could be produced by the bacterial lysys, anyway isn´t your case. You have to recover your protein of the periplasmic space,by means of an osmotic shock, first you have swollen bacteria, and after you plasmolize with an hypotonic solution, liberating your protein and the rest of bacteria remains as esferoplast .So you have to follow some steps, choice an special strain with good ability to avoid S_S intermoleculars as Origami or Rosetta-gami, (I think) and after give a very light induction to let processing of signal peptide at the same time that it¨s produced because if you induce strongly the signal peptidase machinery is unable to process all produced protein, and no processed protein accumulates in citoplasm. Light induction could be done with low inducer conc. low temperature and using lactose in cases that bacteria isn´t lac minus. adieu

William31
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thank you very much for your

thank you very much for your reply.

I will have a look on origami strain.

thanks again

RINKY PARAKRA
RINKY PARAKRA's picture
scfv expression problem

scFvs. I am expressing a scfv antibody (fusion protein) and we have decided to use the vector pET22b which has a pelb sequence for periplasmic translocation. The host cells are bl21De3.I have not added ATG codon at the start as the ATG in the pleb sequence would initiate the protein synthesis.=>pelb_peptideA_linker_scFv_his tagI am seeing the desired band size of protein of interest in the periplasmic fraction. Unfortunately,I am not getting any bands after my pull-down Ni-NTA assay in any of the fractions. Have I a good chance to get the correct folding?I hope the experienced scFv people would have some suggestions.Many thanks.