Protease problems
Yeast secretory pathway endoproteases
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 several yeast secretory pathway endoproteases that may be problematic:
- Kex2 Required for alpha-factor cleavage
- 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.
- 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>
- 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.
- 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.
- Yps6
- Uniprot says: Cleaves proteins C-terminally to mono- and paired-basic residues.
- Yps7
- Uniprot says: Belongs to the peptidase A1 family.
Yapsins (YpsN) proteases are apparently important for cell wall integrity <ref>http://www.ncbi.nlm.nih.gov/pubmed/16087741</ref>.
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>.
We can't get rid of Kex2, but we can potentially
Resources
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>.
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 SLEKREAEA
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 ALNEINQFYQKFPQYLQYLYQGPIVLNPWDQVKRNAVPIT 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 NLYQRRPAIAINNPYVPRTYYANPAVVRPHAQIPQRQYLP NSHPPTVVRRPNLHPSFIAIPPKKIQDKIIIPTINTIATV EPTPAPATEPTVDSVVTPEAFSESIITSTPETTTVAVTPP TA
Fam20C
Six RR sites!
MKMMLVRRFRVLILMVFLVACALHIALDLLPRLERRGARP SGEPGCSCAQPAAEVAAPGWAQVRGRPGEPPAASSAAGDA GWPNKHTLRILQDFSSDPSSNLSSHSLEKLPPAAEPAERA LRGRDPGALRPHDPAHRPLLRDPGPRRSESPPGPGGDASL LARLFEHPLYRVAVPPLTEEDVLFNVNSDTRLSPKAAENP DWPHAGAEGAEFLSPGEAAVDSYPNWLKFHIGINRYELYS RHNPAIEALLHDLSSQRITSVAMKSGGTQLKLIMTFQNYG QALFKPMKQTREQETPPDFFYFSDYERHNAEIAAFHLDRI LDFRRVPPVAGRMVNMTKEIRDVTRDKKLWRTFFISPANN ICFYGECSYYCSTEHALCGKPDQIEGSLAAFLPDLSLAKR KTWRNPWRRSYHKRKKAEWEVDPDYCEEVKQTPPYDSSHR ILDVMDMTIFDFLMGNMDRHHYETFEKFGNETFIIHLDNG RGFGKYSHDELSILVPLQQCCRIRKSTYLRLQLLAKEEYK LSLLMAESLRGDQVAPVLYQPHLEALDRRLRVVLKAVRDC VERNGLHSVVDDDLDTEHRAASAR
References
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