Functional Proteins from a Random-Sequence Library.

 

Abstract

Functional primordial proteins presumably originated from random sequences, but it is not known how frequently functional, or even folded, proteins occur in collections of random sequences. Here we have used in vitro selection of messenger RNA displayed proteins, in which each protein is covalently linked through its carboxy terminus to the 3′ end of its encoding mRNA1, to sample a large number of distinct random sequences. Starting from a library of 6 × 1012 proteins each containing 80 contiguous random amino acids, we selected functional proteins by enriching for those that bind to ATP. This selection yielded four new ATP-binding proteins that appear to be unrelated to each other or to anything found in the current databases of biological proteins. The frequency of occurrence of functional proteins in random-sequence libraries appears to be similar to that observed for equivalent RNA libraries2,3.

 

Acknowledgements

We thank members of the Szostak laboratory and especially D. Wilson, G. Cho, G. Short, J. Pollard, G. Zimmermann, R. Liu, J. Urbach and R. Larralde-Ridaura for their helpful advice. This work was supported in part by the NASA Astrobiology Institute and the NIH. J.W.S. is an investigator at the Howard Hughes Medical Institute.

Author information

Affiliations

  1. Howard Hughes Medical Institute, and Department of Molecular Biology, Massachusetts General Hospital, Boston, 02114, Massachusetts, USA

    Anthony D. Keefe & Jack W. Szostak

Corresponding author

Correspondence to Jack W. Szostak.

Supplementary information

Generalized Random Library sequence (DNA)

TTCTAATACGACTCACTATAGGGACAATTACTATTTACAATTACAATGGACTACAAAGACGACGACGATAAGAAGACTYACTGZ(XYZ)18YACTGZ(XYZ)18YACTGZ(XYZ)18YACTGZ(XYZ)18YACTGGTCAGCGAGCTGCCATCATCATCATCATCATATGGGAATGTCTGGATCT

Average nucleotide composition of random parts of Random Library

  • XYZ

    A35330

    T202922

    G272149

    C181729

    (%)

Generalized Random Library sequence (Protein)

MDYKDDDDKKT(Random)81WSASCHHHHHHMGMSGS

Average amino acid composition of random part of Random Library (Protein)

  • Ala 4.1 Leu 7.4

    Arg 6.8 Lys 5.1

    Asn 7.5 Met 4.5

    Asp 5.5 Phe 2.8

    Cys 4.6 Pro 2.8

    Gln 2.6 Ser 6.6

    Glu 4.0 Thr 5.4

    Gly 5.3 Trp 4.4

    His 3.8 Tyr 5.0

    Ile 4.8 Val 7.1

    (%)

Selected clones from round 8

Family A

  • 08-05MNYKDDDDKKTHWYTNSGFAMTSLRFMMIKWYNWWHDQRHRNIRHHRAMAPRN

    CRIQAITPTHGHDLPQSFEDWRRDYRYNRDKTMAKGYQPWSASCHHHHHHMGMSGS

    08-07MDYKDDDDKKTHWYTNSGFAMTSLRFMMIKWYDWWHDQRHRNIRHHRAMAPRN

    CRIQAITPTHGHDLPQSFEDWRWDYRYNRDKTMAKGYQPWSASCHHHHHHMGMSGS

    08-09MDYKDDDDKKTHWYTNSGFAMTSLRFMMIKWHNWWHDQRHRNIRHHRAMAPRN

    CRIQAITPAHGHDLPQSFEDWRWDYRYNRDKTMAKGYQPWSASCHHHHHHMGMSGS

    08-48MDYKDDDDKKTHWYTNSGFAMTSLRFMMIKWYNWWHDQRHRNIRHHRAMAPRN

    CRIQAITPTHGHDPPQSFEDWRWDYRYNRDKTMAKDYQPWSASCHHHHHHMGMSGS

Family B

  • 08-01MDYKDDDDKKTNCHQKRIYRVKPCVICKVAPRDWWMENRHLRIYTMCKTCFSN

    CINYGDDTYYGHDDWLMYTDCKEFSNTYHNLGRLPDEDRWSASCHHHHHHMGMSGS

    08-04MDYKDDDDKKTNWHQRRIYRVKPCVICKVAPRDWWVENRHLRIYTMCKTCFSN

    CINSGDDTYYGHDDWLMYTDCKEFSNTYHNLGRLPDEDRWSASCHHHHHHMGMSGS

    08-08MDYKDDDDKKTNWHQKRIYRVKPCVICKVAPRDWWVENRHLRIYTMCKTCFSN

    CINNGDDTYYGHDDWLMYTDCKEFSNTYHDLGRLPDEDRWSASCHHHHHHMGMSGS

    08-10MDYKDDDDKKTNWHQKRIYRVKPCVICKVAPRDWWVENRHLRIYTMCKTCFSN

    CINYGDDTYYGHDDWLMYTDCEEFSNTYHNLGRLPDEDRWSASCHHHHHHMGMSGS

    08-12MDYKDDDDKKTNCHQKRIYRVKPCVICKVAPRDWWVENGHLRIYTMCKTCFSN

    CINYGDDTYYGHDDWLMYTDCKEFSNTYHNLDRLPDEDRWSASCHHHHHHMGMSGS

    08-13MDYKDDDDKKTNWHQKRIYRVKPCVICKVAPRDWWVENRHLRIYTMCKTCFSN

    CINYGDDTYYGHDDWLMYTDCKEFSNTYHNLGRLPDEDRWSASCHHHHHHMGMSGS

    08-14MDYKDDDDNKTNWHQKRIYRVKPCVICKVAPRDWWVENKHLRIYTMCKTCFSN

    CINYGDDTYYGHDDWLMYTDCKEFSNTYHNLGRLPDEDRWSASCHHHHHHMGMSGS

    08-15MDYKDDDDKKTNWHQKRIYRVKPCVICKVAPRDWWVENRHLRIYTMCKTCFSN

    CINYGDDTYYGHDDWLMYTDCKEFSNTYHNLGRLPDEDRWSASCHHHHHHMGMSGS

    08-18MDYKDDDDKKTNWHQERIYRVKPCVICKVAPRDWWVENRHLRIYTMCKTCFSN

    CVNYGDDTYYGHDDWLMYTDCKEFSNTYHNLGRLPDEDRWSASCHHHHHHMGMSGS

    08-21MDYKDDDDKKTNWHQKRIYRVKPCVICKVAPRDWWVENRHLRIYTMCKTCFSN

    CINNGDDTYYGHDDWLMYTDCKEFSNTYHNLGRLPDEDRWSASCHHHHHHMGMSGS

    08-23MDYKDDDDKKTNWHQKRIYRVKPCVICKVAPRDWWVENRRLRIYTMCKTCFSN

    CINYGDDTYYGHDDWLMYTDCKEFSNTYHNLGRLPDEDRWSASCHHHHHHMGMSGS

    08-45MDYKDDDDKKTNWHQKRIYRMKPCVICKVAPRDWWVENRHLRIYTMCKTCFSN

    CINYGDDTYYGHDDWLIYTDCKEFSNTYHNLGRLPDEDRWSASCHHHHHHMGMSGS

    08-46MDYKDDDDKKTNWHQKRIYRVKPCVICKVAPRDWWVENRHLRIYTMCKTCFSN

    CINYGDDTYYGHDDWLMYTDCKEFSNTYHNLGRLPDEDRWSASCHHHHHHMGMSGS

    08-47MDYKDDDGKKTNWHQKRIYRVKPCVICKVAPRDWWVENRHLRIYTMCKTCFSN

    CINYGDDTYYGHDDWLMYTDCKEFSNTYHNLGRLPDEDRWSASCHHHHHHMGMSGS

Family C

  • 08-11MDYKDVDDKKTHCDKTVSVDMTFRVRNMKVAKDCWSVVVWTKRSNYFSGRQLH

    CDSWHHYNSRRFGTETKLAYWELPKWKWKINNTHAINIHWSASCHHHHHHMGMSGS

    08-17MDYKDIDDKKTHCDKAVSVDMTFRVRNMKVAKDCWSVVVWTKRSNYFNGRQLH

    CDSWHHYNSRRFGTETKLAYWELPKWKWKINNTHAINIHWSASCHHHHHHMGMSGS

    08-19MDYKDIDDKKTHCDKAVSIDMTFRVRNMKVAKDCWSVVVWTKRSNYFSGRQLH

    SDSWHHYNSRRFGTETKLAYWELPKWKWKINNTHAINIHWSASCHHHHHHMGMSGS

    08-06MDYKDVDDKKTHCDKAVSVDMTFRVRNMKVAKDCWSVVVWTKRSNYFSGRQLH

    CDSWHHYNSRRFGTETKLAYWELPKWKWKINNTHAINIHWSASCHHHHHHMGMSGS

    Family D

    08-20MDYKDDDDKKTYWHALVTYNKTLSYRLATKFTDWWNLDPPRNMQTKVSELNLH

    WLKSGGKGTQKAHSINEISNWVHQHELSDKSMRLHSKVRWSASCHHHHHHMGMSGS

Selected clones from round 18

18-01MDYKDDDDKKTNWQKRIYQVRPCVICKVAPRDWRVENRHLRIYNMCKTCFSNS

VDYGDDTYYGHDDWLMYTDCKEFSNTYHNLGRLPDEDRHWSASCHHHHHHMGMSGS

18-02MDYKDDDDKKTNWQKRVYRARPCVICKVAPRDWRVVNRHLRIYNMCKTCFSNS

INHGDDTYHGHNDWLMYTDCEEFSSTCHNLGRQPDEDRHWSASCHHHHHHMGMSGS

18-03MDYKDDDDKKTYWQKRIYRVRPCVICKVAPRDWRVKNGHLRIYNMCKTCFSNS

IKCGDDTYYGHDDWLIHTDCKDFSNTYLNLGRLPDEERHWSASCHHHHHHMGMSGS

18-04MDYKDDDDKKTNWQKRVYRVRPCVVCKEAPRDWRVKDRHLRIYNMCKTCFSNS

INYGDDTHYGHDDWLMYTDCKGFSNTYHNPSRLPDEDRHWSASCHHHHHHMGMSGS

18-05MDYKDDDDKKTNWQKRIYRVGPCVICKVAPRDWRVENRHLRIYTMCKTCFSNS

IYYGDNTYHGHEDWLMYTDSKEFSNTYHNQGRLPDVDRHWSASCHHHHHHMGMSGS

18-06MDYKDDDDKKTNWQKRIYRVKPCVICKVAPRDWRVENRHLRIYNMCKTCFSNS

INNGDDTYHGHDDWLMYTDCKEFSNTYHNLGRLPDEDRHWSASCHHHHHHMGMSGS

18-07MDYKDDDDKKINWQKRTYRVRPCVICKVAPRDWRVVNRHLRIYNMCKTCFSNS

INYGDDTYYGHDDWLMYTDCKEYSNTYHNLGRLPDEDRHWSASCHHHHHHMGMSGS

18-08MDYKDDDDKKTNWQKRFYRVRPCVICKVAPRDWRVKNGHLRIYNMCKTCFSNS

IKYGDDTYYGHDDWLMYTDCKEFSNTYHNLGRLPNEDRHWSASCHHHHHHMGMSGS

18-09MDYKDDDDKKTNWQKRFYRVKPCVFCKVAPRDWRVENGHLRIYNMCKTCFSNS

LNNGDDTHYGHDDWLMYTDCKEFSNTYHNLGRLPDEDRHWSASCHHHHHHMGMSGS

18-10MDYKDDDDKKTNWQKRIYRVRPCVRCKVAPRDWRVENRHLRIYNMCKTCYSNS

INYGDDTYYGHEDWLLYTDCEEFSNTYHNLGRLPDEDRHWSASCHHHHHHMGMSGS

18-11MDYKDDDDKETSWHKLMYQVRPCVICKVAPRDWRVENRHLRIYTMCKTCFNNS

VNYGDDTHHGHNDWLMYADCNEFTNTCRNLARLPDEDRHWSASCHHHHHHMGMSGS

18-12MDYKDDDDKKTNRQKLIFRVKPCVICKVAPRDWQVENGHLRIYNMCKTCFINS

INNGDDTYHGHDDWLMHTDCTEFSNTYHNLGRLPGEDRHWSASCHHHHHHMGMSGS

18-13MNYKDDDNKKTNWQNRINRVRPCVICKVAPRDWCVKNGHLRIYNMCKSCFSDC

INYGDDTHYGHEDWLMYTDCKEFSNTYHNLGRIPEKDRHWSASCHHHHHHMGMSGS

18-14MATKDDDDKKTNRQKRIFRVKPCVICKVAPRDWRVRNGHLRIYNMCKTCFSNS

INYGDDTYYGHDDRLMYTDCMEFSNTYHNLGKLPDEDRHWSASCHHHHHHMGMSGS

18-15MDYKDDDDKKTNWLKRIYRVKPCVNCKVAPRDWRVKNRHLRIHNMCKTCYSNS

VNYGDDTYYGHDDWLMYTDCEEFSNTYPNLGSLPDEDRHWSASCHHHHHHMGMSGS

18-16MDYKDDDVKKTNWQKRIYRVRPCVICKVAPRDWRVENRHLRIYNMCKTCFSNS

INNGDDTYYGHDDWLMYTDSKEFSYTYHNLGWQPVEDRHWSASCHHHHHHMGMSGS

18-17MDYKDDDDKKTNWQKRTYRVRPCVICKVAPRDWRVKNRHLRIYNMCKTCFSNS

INYGEDTYYGHEDWLMYTDCEEFSKTYHNLGRLPGEDRHWSASCHHHHHHMGMSGS

18-18MDYKDDDDKKTNWQKRIYRVKPCVNCKVAPRDWRVKNRHLRIYNMCKTCYSNS

VNYGDDTYYGHDDWLMYTDCEEFSNSYHNLGRLPDEDRHWSASCHHHHHHMGMSGS

18-19MDYKDDDDKKTNWLKRIYRVRPCVKCKVAPRNWKVKNKHLRIYNMCKTCFNNS

IDIGDDTYHGHDDWLMYADSKEISNTYHNLGRLPNEDKHWSASCHHHHHHMGMSGS

18-20MDYKDDDDKKTNWQKRIYRVGPCVICKVAPRDWRVENGHLRIYNMCKTCFGNS

INNGDDTNFGHDDWLMYTDCKEFSNTYHHLGGLPDEDRHWSASCHHHHHHMGMSGS

18-21MDYKDDDDKKTNWQKRIHRVGPCVICKVAPRDWRVRNRHLRIYNMCKTCFSNS

IKYGDDTYYGHDDWLMYTDCKEFSDTYHNLGRLPDEDRHWSASCHHHHHHMGMSGS

18-22MDYKDDDDKKTNRQKRIYRVRPCVICKVAPRDWRVENRHLRVYNMCKTCFSNS

IHYGDDTYHGHDDWLLHTDCKEFSNTYHQLGRMPDEARHWSASCHHHHHHMGMSGS

18-23MDYKDYDDKKTNWQKRICRVKPCVICKVAPRDWRVKNRHLRIYNMCKTCFSNS

IKYGDDTYYGHDDWLMNTDCKEFSNTYHNLGRLPDEDRHWSASCHHHHHHMGMSGS

18-24MDYKDDDDKKTNRQERLCRVRPCVFCKVAPRDWRVENKHLRIYNMCKTCFSNS

IKYGDDTYHGHDDWLMYTDCKEFSNTYHNLDRLPDEDRHWSASCHHHHHHMGMSGS

18-25MDYKDDDDKKTNWQKRIYRVRPCVICKVAPRDWRVKNRHLRIYNMCKSCFSNS

INNGDDTYYGHDDWLMYTNCEEFSSTYHNLGRLPDEDRHWSASCHHHHHHMGMSGS

18-26MDYKDDDDKKTNRQKRLFRVRPCVICKVAPRDWRVENGHLRIYNMCKTCFSNS

ISNGDDTYFGHEDRLIYSDCKEFSKTNHNPGRLPDVDKHWSASCHHHHHHMGMSGS

18-27MDYKDDDDKKTNWQKRNYWVRPCVICKEAPRDWRVENRHLRIYNMCKTCFSNS

IKSGDDTYYGHDDWLMYTDCKEFSDRYHNLARLPYEDRHWSASCHHHHHHMGMSGS

18-29MDYKDDDDKKTNWQKRNYRVRPCVICRVAPRDWRVKNGHLRIYNMCKTCFSNS

INYGDDTYYGHDDWLMYTDSEEFSNTYHNLDRLPDGDRHWSASCHHHHHHMGMSGS

18-30MDYKDDDDKKTNWQKPIYRVRPCVICKVAPRDWRVKNRNLRIYNMCKTCFSDS

IKYGDDTFHGHDDRLMFTDSKEFSNTYHDQGRQPDEDRHWSASCHHHHHHMGMSGS

18-31MDYKDDDDKKTNWQKRIYRVRPCVICKEAPRDWRVKNGHLRIYNMCKTCFSNS

INYGDDTYHGHDDWLIYKDCKEFSNMYHNLGRLPDEDRHWSASCHHHHHHMGMSGS

18-32MDYKDDDDKKTNWQKRIYRVKPCVICKVAPRNWRVENRHLRIYNMCKTCYSNS

INYGDDTYHGHDDWLMYTGCKEFSNTYHNLGRLPDEVRHWSASCHHHHHHMGMSGS

18-33MNYKDDDDKKTNWQKHILRVRPCVVCKVAPRDWRVKNKHLRIYNMCKTCFSNS

INCGDDTHYGHKDWLIYTDCKESSKTYHDLGRLPDEDRHWSASCHHHHHHMGMSGS

18-34MDYKGDDDKKTNRQKRIYRARPCVICKVAPRDWRVEKRHLRIYNMCKTCYNNS

INYEDDTYHGHDDLGMYTDCKEFSNTYHDLGRLPDEDKHWSASCHHHHHHMGMSGS

18-37MDYKDDDDKKPNWLKRNHRVRPCMICKVAPRDWRVENGHLRIYTMCKTCFGNS

INYGDDTHHGHEDLWMNTDCKEYSYAYHNLGRLPHEDRHWSASCHHHHHHMGMSGS

18-38MDYKDDDDKKTNWKKRIYQVRPCVNCKVAPRDWRVENRHLRVYNMCRTCFSNS

INYGDDTFYGHDDWLLHTDCKQFSNTYHNLGRPPDEDRHWSASCHHHHHHMGMSGS

18-39MDYKDDDDKKTNWLKRIYRVRPCVVCKVAPRDWRLKNGHLRIYNMCKTCFSNS

TNNGDDTYYGHDDWLMNTDCKEFSNSYHNLGRLPDEDRAWSASCHHHHHHMGMSGS

18-41MDNKDDDDKKTNWQKCFYRVRPCVVCKAAPRDWRVENRRLRIYNMCKTCYSNS

INFGDDTHYGHDDWLMYSDSKEFSNTYHNLGRPPDEERHWSASCHHHHHHMGMSGS

18-44MDYKDDDDKKTNWQKRIYQMKPCVVCKVAPRDWRVKNRHLRIYNMCKTCFSNS

INYGDDSYYGHDDWLMNTNSKEFYNTYHNLGRLSDADRHWSASCHHHHHHMGMSGS

18-46MDYKDDDDKKTNRQKRIYRVRPCVICKVAPQDWRVENRRLRIYNMCKTCFSNS

INYGDDTHYGHVDWLMDMDSKEFSNTYHNLGRLPVEDRHWSASCHHHHHHMGMSGS

18-47MDYKDDDDKKTNWQKRIYRVRPCVVCKEAPRDWRMVDRHLRIYNMCKTCFSNS

NNYGDDTYYGHDDRLLYTDCKESSNTYHNPGGLPDEDRHWSASCHHHHHHMGMSGS

18-48MDYKDDDDKKTNWQKRIYRVRPCVICKVAPRDWRVENRHLRIYNMCKTCFSNS

IKYGDDTYHGHDDWLMNTDCKVFSNTYHNLGRLPDEDRHWSASCHHHHHHMGMSGS

18-51MDYKDDDDKKANWQKHIYRVRPCVICKVAPRDWWMENGHLRIYTMCKTCFSNS

INNGDDTYHGHEDWLMYKDCKEFSSTYHNLGRLPVEDRHWSASCHHHHHHMGMSGS

18-52MDYKDDDYKKTNWQKPIFRARPCVKCKVAPRDWWVENRHLRIYNMCKTCFNNS

INYGDDTYYGHDDWLVYTDCKEFSNTYHNLGRLPDEDRHWSASCHHHHHHMGMSGS

18-54MDYKDDDDKKTNWQKRIYRVRPCVICKVAPRDWRVENRHLRIYNMCKTCFSNS

INHGDDTYFGHDDWLMYTDRKEFSNTYYNLGRLPGEDRHWSASCHHHHHHMGMSGS

18-56MDYKDDDDKKTNWQKHIYRVRPCVRCKVAPRDWRVENGHLRIYNMCKTCFSNS

INYGDDTNYGHDDWPLYTDSKEFSNTYHNLDRPPDEDRHWSASCHHHHHHMGMSGS

18-57MDYKDDNDKMTNRQKRIYRVRPCVVCKVAPRDWRVENRHLRIYNMCKTCFSDS

IKYRDDTHHGHDDWLMYTDCMEFSNTYHNLGWLPDEDRHWSASCHHHHHHMGMSGS

18-60MDYKDDDDKKTNWLKRNYRVKPCVNCKVAPRDWRVKNRHLRIYNMCKTCYSNS

INYGDDTYYGHDDWLMYTDCEEFSNTYHNRGRLPDEDRHWSASCHHHHHHMGMSGS

18-61MDYKDDDDKKTNWRKRIYRVRPCVICKVAPRDWRVEDSHLRIYNMCKTCFSNS

INYGDDTYFGHEDWLMYTDCKEFSNTYHNLDRLPDENRHWSASCHHHHHHMGMSGS

18-62MDYKDDDDKKTNWQKRIYRVRPCVKCKVAPRDWRVENSHLRIYNMCKTCFSNS

INYGDDTHYGHVDWVMYTDCMKFSNTYHNLSRLPDENRHWSASCHHHHHHMGMSGS

18-63MDYKDDDDKKAYWQERIYRVKPCVICKVAPRDWRVKNRHLRIYNMCKSCFSNS

IIYGDDTYHGHDDWLMYTDCKEFFNTYHNLGRLPDEDRHWSASCHHHHHHMGMSGS

18-64MDYKDDDDKKTYWQKRIYRVKPCVICKEAPRDWRVKNRHLRIYNMCKTCFSNS

VNIGDDTYHGHDDWLDEYDCKEFSNTCHNLGRLPGEDKHWSASCHHHHHHMGMSGS

18-65MDYKDDDDKKTYWQKRIYRVRPCVVCKVAPRDWRVENRHLRIYNMCKTCFSNS

INYGDDTHYGHDDWLMNTDCKEFSNTYHNPGKLPDEDRHWSASCHHHHHHMGMSGS

18-66MDYKDDDDKKTNWQKSIYREKPCVVCKVAPRDWRVENGHLRIYNMCKTCYSNS

INYGDDTYYGHDDWLMYKDCKEFSNTYHYQGRLPEEDRHWSASCHHHHHHMGMSGS

18-67MDYKDDDDKKTNWQKRIYRVRPCVICKVAPRDWRVENRHLRIYNMCKTCFSNS

INNGDDTYHGHDDWLLYTDRKEFSNTYHNLGRLPDEDRHWSASCHHHHHHMGMSGS

18-68MDYKDDDDKKTNWQKRIYRVRPCVICKVAPRDWRVENRHLRIYNMCKTCFSNS

INYGDDTFYGHDDWLMYTDSKEFSNTYHNLGRLPDEDRHWSASCHHHHHHMGMSGS

18-72MDYKDDDDKKTIRQKRIYRVRPCVNCKVAPRDWRVENRHLRIYNMCKTCFSNS

INYRDDTYYGHDDWLIYTDCKEFSNTYHNLGRLPDKDRHWSASCHHHHHHMGMSGS

Fuller description of protocol for a round of selection:

In vitro selection and amplification

RNA was produced from the DNA library with T7 RNA polymerase and mRNA-displayed proteins were generated as previously described8,9. The linker, which connects the RNA 3’-terminus to the protein C-terminus, had the following sequence: (dA)21(triethylene glycol phosphate ester)3dAdCdCPuromycin (made using reagents from Glen Research, Sterling, VA). A 10 ml translation (400 nM template, Red Nova Rabbit Reticulocyte Lysate (Novagen, Madison, WI), according to the manufacturer’s instructions with 85 mM additional KCl, 0.85 mM additional Mg(OAc)2 and 25 nM 35S-methionine) incubated at 30°C for one hour yielded 7×1013 mRNA-displayed proteins after high-salt incubation (600 mM KCl, 25 mM MgCl2). The translation mixture was then diluted ten-fold into oligo(dT)cellulose binding buffer (1 M KCl, 100 mM Tris(hydroxymethyl) amino methane, 0.25% w/v Triton X-100, pH 8.0) and this mixture was incubated with 2 mg/ml oligo(dT)cellulose (Pharmacia, Piscataway, NJ) for fifteen minutes at 4°C with rotation. The oligo(dT)cellulose was washed on a chromatography column (Bio-Rad, Hercules, CA) with the same oligo(dT)cellulose binding buffer and then eluted with deionized water. The eluate was mixed with 2x Ni-NTA binding buffer (1x is 6 M guanidinium chloride, 0.5 M NaCl, 100 mM sodium phosphate, 10 mM Tris(hydroxymethyl)amino methane, 10 mM 2-mercaptoethanol, 0.25% w/v Triton X-100, pH 8.0) and then incubated with Ni-NTA agarose (Qiagen, Valencia, CA) for one hour at 4°C with rotation. The Ni-NTA agarose was then washed with Ni-NTA first wash buffer (8 M urea, 0.5 M NaCl, 100 mM sodium phosphate, 10 mM Tris(hydroxymethyl)amino methane, 10 mM 2-mercaptoethanol, 0.25% w/v Triton X-100, pH 6.3) and then with a gradient of increasing amounts of Ni-NTA second wash buffer (0.5 M NaCl, 10 mM Tris(hydroxymethyl)amino methane, 10 mM 2-mercaptoethanol, 0.25% w/v Triton X-100, pH 8.0), and then was eluted with Ni-NTA elution buffer (0.25 M imidazole, 0.5 M NaCl, 10 mM Tris(hydroxymethyl) amino methane, 10 mM 2-mercaptoethanol, 0.25% w/v Triton X-100, pH 8.0) for one hour at 4°C with rotation. EDTA was added to the eluate to give a concentration of 5 mM. The buffer was exchanged into Reverse Transcription buffer (50 mM Tris(hydroxymethyl) amino methane, 75 mM KCl, 3 mM MgCl2 pH 8.3) on a gel filtration column (Pharmacia, Piscataway, NJ). The mRNA-displayed proteins were then reverse transcribed with Superscript II (Gibco BRL, Rockville, MD) at 42°C for 30 minutes without a heat denaturation step. This sample was then exchanged into selection binding buffer (400 mM KCl, 20 mM HEPES, 4 mM MgCl2, 0.1mM EDTA, 2 mM glutathione, 1 mM glutathione disulfide, 0.25% w/v Triton X-100, pH 7.4) on a gel filtration column (Pharmacia, Piscataway, NJ), and then incubated with 100 µl of ATP-agarose (ATP attached via C8, 9 atom linker, cyanogen bromide activated cross-linked 4% beaded agarose (Sigma, St. Louis, MO)) at 4°C for one hour on a chromatography column (Bio-Rad, Hercules, CA). The column was then washed with 50 column volumes of selection binding buffer at 4°C for one hour and eluted with 12 column volumes of selection elution buffer (as selection binding buffer, but with an additional 5 mM ATP and 4.8 mM MgCl2, pH readjusted to 7.4) at 4°C for one hour. The eluted fraction was then brought to 0.1 M in NaOH, hydrolyzed at 90°C for 10 minutes, exchanged into deionized water on two successive gel filtration columns and amplified using PCR. Every round was assayed by SDS-PAGE to ensure that mRNA degradation had not occurred, and by scintillation counting of the 35S-methionine labelled proteins to measure the efficiencies of the various steps. These data were then used to determine the number of purified individual protein sequences introduced into the round one selection step as 6×1012 based on the proportion of total methionine incorporated into the mRNA-displayed proteins, and the efficiency of each of the subsequent purification steps.

This procedure was repeated for 18 rounds except that in rounds 10, 11 and 12 the PCR amplification was substituted by a mutagenic PCR amplification with an average mutagenic rate of 3.7% at the amino acid level. In rounds 14, 15 and 16 the amplification cycles were preceded by two ATP-agarose selection steps, and in rounds 17 and 18 the amplification cycles were preceded by three ATP-agarose selection steps. With successive selection steps the eluted fraction was exchanged into deionized water on a gel filtration column and purified on a denaturing Ni-NTA column and reverse transcribed as described above before being incubated with the ATP-agarose for the subsequent selection step. Also, the volume of the translation reaction was reduced to 1 ml except for round 1 in which it was 10 ml, and rounds 2, 10, 11 and 12 in which it was 5 ml. In the rounds of selection preceding the initial mutagenic amplification, incubation with a butyl agarose pre-column (Sigma, St. Louis, MO) was employed with the flowthrough being used for incubation with the ATP-affinity column.

K d by spin-filtration

Purified MBP-fusion proteins were exchanged into selection binding buffer by gel filtration and mixed with [α32P-ATP. These samples were incubated at 4°C for 30 minutes. 200 µl samples were then placed into Microcon-30 spin ultrafiltration devices (Millipore, Bedford, MA) and spun at 10,000g for 30 seconds with this filtrate being discarded, spinning at 10,000g for a further 45 seconds yielded a subsequent filtrate which, along with the unfiltered sample, were assayed by scintillation counting. This method was adapted from14. In competition experiments, the competitor was added after the incubation step, and then the solution was incubated for an extra 30 minutes at 4°C. Data were treated as above (Kd by Equilibrium Dialysis) and according to the method of Wang and von Hippel15 to measure the stoichiometry of the interaction.

K d by equilibrium dialysis

Purified MBP-fusion proteins were exchanged into selection binding buffer by gel filtration and 150 µl aliquots were placed on one side of a 14-16 kDa MWCO dialysis membrane in a Hoefer Scientific Instruments model EMD101B. Equal volumes of the same buffer containing diluted [α32P-ATP were placed on the other side of the membrane. After 24 hours at 4°C, samples were removed from each side of the membrane and assayed by scintillation counting. These data were fitted to y=c(x/(x+ Kd)), where x is the protein concentration, y the proportion of counts bound by the protein and c is the proportion of counts that are able to be bound by the protein, by an iterative algorithm (Deltagraph) to give the Kd. Competition experiments against unlabelled ATP or ATP analogues were performed similarly.

Back titration of hot ATP against cold ATP to give stoichiometry of interaction

This data was fitted to y = b+c((ANK+PK+1)

-((ANK+PK+1)2-(4ANPK2))0.5)/2PK

  • b is proportion of counts not passing through membrane, an iteratively fitted constant

    c is proportion of counts bindable, an iteratively fitted constant

    A is the ATP concentration

    K is the association constant

    P is the active protein concentration

    N is the stoichiometry, an iteratively fitted constant

References :

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Link collected : https://www.nature.com/articles/35070613