Difference between revisions of "Plasmid design"

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We’ve chosen EcoRI and NotI as our restriction enzymes, because they cut only in the right places on our plasmid.


Title: Protease problems
The following PDFs map the pPIC3.5K plasmid with each of the bovine casein genes inserted.


= Yeast secretory pathway endoproteases =
[[:File:pPIC35K-Ost1-Alpha-AS1-sequence.pdf]]


The small size and limited secondary and tertiary structure in our target proteins may make them more prone to unwanted degredation by proteases in the yeast secretory pathway. Research turns up three yeast secretory pathway endoproteases that may be problematic:
[[:File:pPIC35K-Ost1-Alpha-AS2-sequence.pdf]]


*[http://www.uniprot.org/uniprot/P13134 Kex2] Required for alpha-factor cleavage
[[:File:pPIC35K-Ost1-Beta-sequence.pdf]]
** Seems like it primarily cleaves Lys-Arg (KR) and Arg-Arg (RR) but can also cleave other Arg-containing sites.
** Uniprot says: Cleavage of -Lys-Arg-|-Xaa- and -Arg-Arg-|-Xaa- bonds to process yeast alpha-factor pheromone and killer toxin precursors.
*[http://www.uniprot.org/uniprot/P32329 Yps1] (also known as Yap3)
** "The cleavage site of this enzyme appeared identical to that of the KEX2-encoded endopeptidase." <ref>http://www.ncbi.nlm.nih.gov/pubmed/2183521</ref>
*[http://www.uniprot.org/uniprot/P53379 Yps2] (also known as Mkc7)
**Uniprot says: Hydrolyzes various precursor proteins with Arg or Lys in P1, and commonly Arg or Lys also in P2. The P3 amino acid is usually non-polar, but otherwise additional basic amino acids are favorable in both non-prime and prime positions.
*[http://www.uniprot.org/uniprot/Q12303 Yps3] <ref>http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1220171/pdf/10191273.pdf</ref>
**Uniprot says: Cleaves proteins C-terminally to mono- and paired-basic residues.
*[http://www.uniprot.org/uniprot/P40583 Yps6]  
**Uniprot says: Cleaves proteins C-terminally to mono- and paired-basic residues.
*[http://www.uniprot.org/uniprot/Q06325 Yps7]
**Uniprot says: Belongs to the [http://www.uniprot.org/uniprot/?query=family:%22peptidase+A1+family%22 peptidase A1 family].


[[:File:pPIC35K-Ost1-Kappa-sequence.pdf]]


Yapsins (YpsN) proteases are apparently important for cell wall integrity <ref>http://www.ncbi.nlm.nih.gov/pubmed/16087741</ref>.
We then used Benchling to optimize the codons for expression in the yeast Pichia pastori.


People have used a strain lacking Yap3 and Mkc7 genes to express proteolytically sensitive short (41 aa) peptides in S. cerevisiae <ref>http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1219280/pdf/9494104.pdf</ref>.
= Protease problems =


== Resources ==
See the [[protease problems]] page.
 
[http://books.google.com/books?id=XBuHJLFRjRkC&pg=PA176&lpg=PA176&dq=s+cerevisiae+Yps2&source=bl&ots=Bd1Ot-XT0N&sig=TgNq71uUo_SJYlypa1WFCi_LSM8&hl=en&sa=X&ei=fIrxU5etCIrroATR9wE&ved=0CEEQ6AEwBA#v=onepage&q=s%20cerevisiae%20Yps2&f=false Handbook of Proteolytic Enzymes]
 
== Yps1 / Yap3 ==
 
One of the yapsins. The yapsins are apparently important for cell wall integrityt.
 
"The cleavage site of this enzyme appeared identical to that of the KEX2-encoded endopeptidase." and "Strains disrupted in YAP3 are both viable and able to process the mating factor alpha precursor." <ref>http://www.ncbi.nlm.nih.gov/pubmed/2183521</ref>
 
== Kex2 ==
 
Kex2 is a high-specificity membrane-bound endoprotease involved in cleaving alpha-factor in the secretory pathway.
 
Kex2 potentially cleaves at Lys-Arg|, Arg-Arg|, Pro-Arg|, Ala-Arg| and Thr-Arg| but seems to prefer Lys-Arg| <ref>http://www.ncbi.nlm.nih.gov/pubmed/1736307</ref>.
 
[https://en.wikipedia.org/wiki/Kexin Wikipedia says] that it cleaves Lys-Arg and Arg-Arg bonds.
 
Kex2 potentially cleaves at Arg|, Arg|Lys, Pro-Arg| <ref>http://www.ncbi.nlm.nih.gov/pubmed/7819327</ref>.
 
= Target proteins =
 
Kex2 protease cleaves primarily at KR or RR sites. It is responsible for cleaving off the alpha-factor secretion signal.
 
== Full alpha-factor yeast secretion signal==
 
Has KR site near C-terminal end (as it should)
 
MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIG
YSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGV
SLE'''KR'''EAEA
 
== CSN1S1 bovine ==
 
No problematic sites
 
RPKHPIKHQGLPQEVLNENLLRFFVAPFPEVFGKEKVNEL
SKDIGSESTEDQAMEDIKQMEAESISSSEEIVPNSVEQKH
IQKDDVPSERYLGYLEQLLRLKKYKVPQLEIVPNSAEERL
HSMKEGIHAQQKEPMIGVNQELAYFYPELFRQFYQLDAYP
SGAWYYVPLGTQYTDAPSFSDIPNPIGSENSGKTTMPLW
 
== CSN1S1 human ==
 
No problematic sites
 
RPKHPIKHQGLPQEVLNENLLRFFVAPFPEVFGKEKVNEL
SKDIGSESTEDQAMEDIKQMEAESISSSEEIVPNSVEQKH
IQKDDVPSERYLGYLEQLLRLKKYKVPQLEIVPNSAEERL
HSMKEGIHAQQKEPMIGVNQELAYFYPELFRQFYQLDAYP
SGAWYYVPLGTQYTDAPSFSDIPNPIGSENSGKTTMPLW
 
== CSN1S2 bovine ==
 
One KR site!
 
KNTMEHVSSSEESIISQETYKQEKNMAINPSKENLCSTFC
KEVVRNANEEEYSIGSSSEESAEVATEEVKITVDDKHYQK
ALNEINQFYQKFPQYLQYLYQGPIVLNPWDQV'''KR'''NAVPIT
PTLNREQLSTSEENSKKTVDMESTEVFTKKTKLTEEEKNR
LNFLKKISQRYQKFALPQYLKTVYQHQKAMKPWIQPKTKV
IPYVRYL
 
== CSN1S2 human ==
 
This gene doesn't exist in humans.
 
== CSN2 bovine A2 variant ==
 
No problematic sites.
 
RELEELNVPGEIVESLSSSEESITRINKKIEKFQSEEQQQ
TEDELQDKIHPFAQTQSLVYPFPGPIPNSLPQNIPPLTQT
PVVVPPFLQPEVMGVSKVKEAMAPKHKEMPFPKYPVEPFT
ESQSLTLTDVENLHLPLPLLQSWMHQPHQPLPPTVMFPPQ
SVLSLSQSKVLPVPQKAVPYPQRDMPIQAFLLYQEPVLGP
VRGPFPIIV
 
== CSN2 bovine B variant ==
 
No problematic sites.
 
RELEELNVPGEIVESLSSSEESITRINKKIEKFQSEEQQQ
TEDELQDKIHPFAQTQSLVYPFPGPIHNSLPQNIPPLTQT
PVVVPPFLQPEVMGVSKVKEAMAPKHKEMPFPKYPVEPFT
ERQSLTLTDVENLHLPLPLLQSWMHQPHQPLPPTVMFPPQ
SVLSLSQSKVLPVPQKAVPYPQRDMPIQAFLLYQEPVLGP
VRGPFPIIV
 
== CSN2 Human ==
 
No problematic sites.
 
RETIESLSSSEESITEYKQKVEKVKHEDQQQGEDEHQDKI
YPSFQPQPLIYPFVEPIPYGFLPQNILPLAQPAVVLPVPQ
PEIMEVPKAKDTVYTKGRVMPVLKSPTIPFFDPQIPKLTD
LENLHLPLPLLQPLMQQVPQPIPQTLALPPQPLWSVPQPK
VLPIPQQVVPYPQRAVPVQALLLNQELLLNPTHQIYPVTQ
PLAPVHNPISV
 
== CSN3 bovine B variant ==
 
No problematic sites.
 
QEQNQEQPIRCEKDERFFSDKIAKYIPIQYVLSRYPSYGL
NYYQQKPVALINNQFLPYPYYAKPAAVRSPAQILQWQVLS
NTVPAKSCQAQPTTMARHPHPHLSFMAIPPKKNQDKTEIP
TINTIASGEPTSTPTIEAVESTVATLEASPEVIESPPEIN
TVQVTSTAV
 
== CSN3 human ==
 
Two RR sites!
 
EVQNQKQPACHENDERPFYQKTAPYVPMYYVPNSYPYYGT
NLYQ'''RR'''PAIAINNPYVPRTYYANPAVVRPHAQIPQRQYLP
NSHPPTVV'''RR'''PNLHPSFIAIPPKKIQDKIIIPTINTIATV
EPTPAPATEPTVDSVVTPEAFSESIITSTPETTTVAVTPP
TA
 
== Fam20C ==
 
Six RR sites!
 
MKMMLV'''RR'''FRVLILMVFLVACALHIALDLLPRLE'''RR'''GARP
SGEPGCSCAQPAAEVAAPGWAQVRGRPGEPPAASSAAGDA
GWPNKHTLRILQDFSSDPSSNLSSHSLEKLPPAAEPAERA
LRGRDPGALRPHDPAHRPLLRDPGP'''RR'''SESPPGPGGDASL
LARLFEHPLYRVAVPPLTEEDVLFNVNSDTRLSPKAAENP
DWPHAGAEGAEFLSPGEAAVDSYPNWLKFHIGINRYELYS
RHNPAIEALLHDLSSQRITSVAMKSGGTQLKLIMTFQNYG
QALFKPMKQTREQETPPDFFYFSDYERHNAEIAAFHLDRI
LDF'''RR'''VPPVAGRMVNMTKEIRDVTRDKKLWRTFFISPANN
ICFYGECSYYCSTEHALCGKPDQIEGSLAAFLPDLSLAKR
KTWRNPW'''RR'''SYHKRKKAEWEVDPDYCEEVKQTPPYDSSHR
ILDVMDMTIFDFLMGNMDRHHYETFEKFGNETFIIHLDNG
RGFGKYSHDELSILVPLQQCCRIRKSTYLRLQLLAKEEYK
LSLLMAESLRGDQVAPVLYQPHLEALD'''RR'''LRVVLKAVRDC
VERNGLHSVVDDDLDTEHRAASAR
 
= References =
 
<references/>

Latest revision as of 04:12, 27 September 2022

We’ve chosen EcoRI and NotI as our restriction enzymes, because they cut only in the right places on our plasmid.

The following PDFs map the pPIC3.5K plasmid with each of the bovine casein genes inserted.

File:pPIC35K-Ost1-Alpha-AS1-sequence.pdf

File:pPIC35K-Ost1-Alpha-AS2-sequence.pdf

File:pPIC35K-Ost1-Beta-sequence.pdf

File:pPIC35K-Ost1-Kappa-sequence.pdf

We then used Benchling to optimize the codons for expression in the yeast Pichia pastori.

Protease problems

See the protease problems page.

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