Plasmid design

Title: 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 three 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>.

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

<references/>