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AI-domain for its cognate protease transducer. In contrast, truncated DNA libraries are typically applied to optimize the length
and structure of the linkers connecting functional domains. An
exemplary protocol to engineer dipeptide motifs that can bind
across the P1-P1′ junction without being cleaved is exemplified
based on USER Enzyme-dependent DNA assembly [14].
1. Devise a cloning strategy for a focused DNA library to engineer a competitively autoinhibited protease module taking into
account structural and biochemical data from the existing literature (Fig. 2). For instance, in case of TVMV, the design of
uncleavable P1-P1′ junctions for the AI-domain is based on
substrate mapping data for the related TEV protease which
suggests that proline in the P1′ position prevents cleavage
[15]. It is however unclear to what extent sequences with a
proline in the P1′ position cannot bind or can bind, but cannot
be cleaved by TEV protease. Similarly, it is not clear to what
extent this holds true for other members of the NIa potyvirus
protease family including the NIa protease of TVMV [2], but
this needs to be screened and tested experimentally.
2. Commercially synthesize the gene coding for the autoinhibited protease module of interest (Fig. 2). For TVMV, include a
cleavage site for autoproteolytic processing followed by TVMV
protease, a linker including a thrombin cleavage site, its cognate substrate sequence (which serves as a lead structure for
engineering competitive AI-domains), and an affinity purification tag and insert into pRK793 via NcoI and BamHI.
3. Generate a DNA fragment coding for the N-terminal portion
by means of PCR using Pfu C x DNA polymerase, 1× Pfu C x
reaction buffer, 10 mM dNTPs each, 1 ng template DNA coding for the elementary protease module (step 2), and primers
Primer-For and Primer-C-Ter.
4. Generate a DNA fragment coding for the C-terminal portion
by means of PCR using Pfu C x DNA polymerase, 1× Pfu C x
reaction buffer, 10 mM dNTPs each, 1 ng template DNA coding
Fig. 2 Summary of cloning procedure to engineer autoinhibited protease modules exemplified with the NIa
protease of TVMV. (a) First commercially gene synthesize an autoinhibited protease module that serves as a
template for subsequent engineering attempts. This includes restriction sites NcoI and BamHI flanked by 50 bp
homology regions to enable integration of the synthetic DNA fragment into a target vector, in this case pRK793,
by means of Gibson Assembly. The autoinhibited protease module contains an N-terminal cleavage site for
autocatalytic processing from MBP and a C-terminal cleavage site as the lead for a product-based competitive
inhibitor. The latter is additionally separated by a cleavage site for thrombin and an affinity tag to enable purification of the full-length protease transducer. (b, c) Using suitable primer pairs amplify DNA fragments coding
for the N- and C-terminal portion of TVMV while randomizing the P1-P1′ junction using degenerate codons that
will ultimately yield a dipeptide motif that can bind across the P1-P1′ junction, yet cannot be cleaved.
Recombination of the two DNA fragments is mediated through USER enzyme-dependent ligation. (d) Summary
of the USER enzyme-dependent recombination site including the coding amino acid sequence
Viktor Stein and Kirill Alexandrov
AI-domain for its cognate protease transducer. In contrast, truncated DNA libraries are typically applied to optimize the length
and structure of the linkers connecting functional domains. An
exemplary protocol to engineer dipeptide motifs that can bind
across the P1-P1′ junction without being cleaved is exemplified
based on USER Enzyme-dependent DNA assembly [14].
1. Devise a cloning strategy for a focused DNA library to engineer a competitively autoinhibited protease module taking into
account structural and biochemical data from the existing literature (Fig. 2). For instance, in case of TVMV, the design of
uncleavable P1-P1′ junctions for the AI-domain is based on
substrate mapping data for the related TEV protease which
suggests that proline in the P1′ position prevents cleavage
[15]. It is however unclear to what extent sequences with a
proline in the P1′ position cannot bind or can bind, but cannot
be cleaved by TEV protease. Similarly, it is not clear to what
extent this holds true for other members of the NIa potyvirus
protease family including the NIa protease of TVMV [2], but
this needs to be screened and tested experimentally.
2. Commercially synthesize the gene coding for the autoinhibited protease module of interest (Fig. 2). For TVMV, include a
cleavage site for autoproteolytic processing followed by TVMV
protease, a linker including a thrombin cleavage site, its cognate substrate sequence (which serves as a lead structure for
engineering competitive AI-domains), and an affinity purification tag and insert into pRK793 via NcoI and BamHI.
3. Generate a DNA fragment coding for the N-terminal portion
by means of PCR using Pfu C x DNA polymerase, 1× Pfu C x
reaction buffer, 10 mM dNTPs each, 1 ng template DNA coding for the elementary protease module (step 2), and primers
Primer-For and Primer-C-Ter.
4. Generate a DNA fragment coding for the C-terminal portion
by means of PCR using Pfu C x DNA polymerase, 1× Pfu C x
reaction buffer, 10 mM dNTPs each, 1 ng template DNA coding
Fig. 2 Summary of cloning procedure to engineer autoinhibited protease modules exemplified with the NIa
protease of TVMV. (a) First commercially gene synthesize an autoinhibited protease module that serves as a
template for subsequent engineering attempts. This includes restriction sites NcoI and BamHI flanked by 50 bp
homology regions to enable integration of the synthetic DNA fragment into a target vector, in this case pRK793,
by means of Gibson Assembly. The autoinhibited protease module contains an N-terminal cleavage site for
autocatalytic processing from MBP and a C-terminal cleavage site as the lead for a product-based competitive
inhibitor. The latter is additionally separated by a cleavage site for thrombin and an affinity tag to enable purification of the full-length protease transducer. (b, c) Using suitable primer pairs amplify DNA fragments coding
for the N- and C-terminal portion of TVMV while randomizing the P1-P1′ junction using degenerate codons that
will ultimately yield a dipeptide motif that can bind across the P1-P1′ junction, yet cannot be cleaved.
Recombination of the two DNA fragments is mediated through USER enzyme-dependent ligation. (d) Summary
of the USER enzyme-dependent recombination site including the coding amino acid sequence
Viktor Stein and Kirill Alexandrov