Expressed Protein Ligation without Intein

Article abstract of DOI:10.1021/jacs.0c00252

Proteins with a functionalized C-terminus such as a C-terminal thioester are key to the synthesis of larger proteins via expressed protein ligation. They are usually made by recombinant fusion to intein. Although powerful, the intein fusion approach suffe

Full text of DOI:10.1021/jacs.0c00252

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Article
Expressed Protein Ligation Without Intein
Yuchen Qiao, Ge Yu, Kaci C Kratch, Xiaoyan Aria Wang, Wei Wang,
Sunshine Z Leeuwon, Shiqing Xu, Jared S Morse, and Wenshe R. Liu
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.0c00252 • Publication Date (Web): 26 Mar 2020
Downloaded from pubs.acs.org on March 28, 2020
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Expressed Protein Ligation Without Intein
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Yuchen Qiaoa, Ge Yua, Kaci C. Kratcha, Xiaoyan Aria Wanga, Wesley Wei Wanga, Sunshine Z. Leeu-
wona, Shiqing Xua, Jared S. Morsea, and Wenshe Ray Liua,b,c
*
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aThe Texas A&M Drug Discovery Laboratory, Department of Chemistry, Texas A&M University, College Station, TX 77843,
USA
bDepartment of Biochemistry & Biophysics, Texas A&M University, College Station, TX 77843, USA
cMolecular & Cellular Medicine Department, College of Medicine, Texas A&M University, College Station, TX 77843, USA
*Correspondence should be addressed to Wenshe Ray Liu: wliu@chem.tamu.edu
Supporting Information Placeholder
thioester for further native chemical or expressed protein ligation
(Figure 1A)3-4. Evident by the original publication garnering more
than 2700 citations so far, the advent of native chemical and ex-
pressed protein ligation techniques has revolutionized the protein
and peptide chemistry field. Groundbreaking applications include
the synthesis of a large variety of proteins such as histones, kinases,
and RAS proteins with posttranslational modifications for driving
basic research advances and the production of many proteins or en-
zymes for therapeutic and biotechnological purposes5-12. Although
developed extensively, further technological improvement in pro-
tein ligation is still necessary. The production of a protein thioester
using the intein fusion approach is not guaranteed for a lot of pro-
teins. The stringent requirement for the intein catalysis to generate
a protein thioester prevents the processing of many fusion proteins
that are expressed insolubly and hard to fold13. The C-terminal res-
idue of a targeted protein that is immediate to the intein N-terminus
also significantly impacts the protein splicing efficiency, which
leads to low splicing efficiency for residues such as proline at this
site14-15. The purification of an intein fusion also requires signifi-
cant caution for avoiding premature hydrolysis2, 16. A split intein
may be used to prevent premature hydrolysis but adds more proce-
dural complexity5, 17. Using a protein ligase for expressed protein
ligation resolves some issues related to the intein fusion approach
but requires a specific amino acid sequence context at the ligation
site18. Therefore, a simple method to functionalize a recombinant
protein at its C-terminus for expressed protein ligation that requires
no enzymatic catalysis, can be broadly applied, and maintains high
efficiency in different protein C-terminal sequence contexts is
highly desired. In this work, we report such a method and its appli-
cation in the synthesis of a number of proteins or peptides that can
be used in both basic research and therapy.
ABSTRACT: Proteins with a functionalized C-terminus such as
a C-terminal thioester are key to the synthesis of larger proteins via
expressed protein ligation. They are usually made by recombinant
fusion to intein. Although powerful, the intein fusion approach suf-
fers from premature hydrolysis and low compatibility with dena-
tured conditions. To totally bypass the involvement of an enzyme
for expressed protein ligation, here we showed that a cysteine in a
recombinant protein was chemically activated by a small molecule
cyanylating reagent at its N-side amide for undergoing nucleophilic
acyl substitution with amines including a number of L- and D-
amino acids and hydrazine. The afforded protein hydrazides could
be used further for expressed protein ligation. We demonstrated the
versatility of this activated cysteine-directed protein ligation
(ACPL) approach with the successful synthesis of ubiquitin conju-
gates, ubiquitin-like protein conjugates, histone H2A with a C-ter-
minal posttranslational modification, RNAse H that actively hydro-
lyzed RNA, and exenatide that is a commercial therapeutic peptide.
The technique, which is exceedingly simple but highly useful, ex-
pands to a great extent the synthetic capacity of protein chemistry
and will therefore make a large avenue of new research possible.
■ INTRODUCTION
The native chemical ligation concept was first developed by
Dawson et al. in 1994, in which one protein or peptide with a C-
terminal thioester and the other with a N-terminal cysteine selec-
tively undergo thiol-thioester exchange and then S-to-N acyl trans-
fer to form a larger protein or peptide (Figure 1A)1. Given that a
protein with an N-terminal cysteine can be recombinantly pro-
duced, the development of the concept made it feasible to synthe-
size large proteins with a functionalized N-terminus to include ei-
ther posttranslational or purely chemical modifications. To expand
the synthetic scope of native chemical ligation, a related technique
termed expressed protein ligation in which a recombinant fusion
protein with a C-terminal intein is used to generate a protein thioe-
ster was also developed for the synthesis of a protein with a func-
tionalized C-terminus2. Another notable related technique is pep-
tide hydrazide ligation that uses nitrous acid or acetyl acetone to
convert a chemically stable peptide hydrazide to a peptide acyl az-
ide or a peptide acyl pyrazole respectively and then a peptide
■ RESULTS AND DISCUSSION
The Rationale
Among 20 proteinaceous amino acids, cysteine is the most nu-
cleophilic. Its direct activation for generating a peptide thioester
was previously explored by Kajihara, Otaka, and their coworkers19-
20. To develop a more general, cysteine-based approach that could
be applied to proteins for their C-terminal functionalization, we fol-
lowed a century old industrial chemical process, leather tanning by
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cyanides. Cyanide salts that reduce disulfide bonds in proteins were
used in the early 20th century to treat animal hides and wools21-22
were synthesized using the standard amidation approach as con-
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trols. As shown in the 1H NMR spectra (Figure S1), no diastereo-
meric CH3 peak was observed for the ligated product indicating that
this reaction did not lead to the racemization of the amino acid be-
fore cysteine.
During the process, a cyanide covalently attached to a protein cys-
teine to form a thiocyanate that underwent reversible intramolecu-
lar addition with the cysteine N-amide to generate a 1-acyl-2-imi-
nothiazolidine intermediate. The amide bond in this intermediate
was significantly weakened in comparison to a regular protein am-
ide and therefore slowly hydrolyzed to split the protein (Figure
1B)23-24. Early protein chemists exploited this reaction for mapping
protein sequences and replaced cyanide salts with other cyanylat-
ing reagents such as 2-nitro-5-thiocyanatobenzoic acid (NTCB)
that transfers the cyano group directly to a reduced protein cysteine
for avoiding the formation of highly toxic cyanide wastes25-26. Ac-
cording to this reaction mechanism, providing a strongly nucleo-
philic amine in the reaction will trigger nucleophilic acyl substitu-
tion with the 1-acyl-2-iminothiazolidine intermediate to replace 2-
iminothiazolidine and potentially circumvent the hydrolysis pro-
cess. The afforded small molecule amine-ligated product can be
used for a further protein ligation process. Since this proposed ex-
pressed protein ligation that we termed as activated cysteine-based
protein ligation (ACPL) doesn’t involve an enzyme and is purely
chemically based, it can be highly controllable, selective, and ver-
satile such as functioning for proteins both soluble and insoluble.
Versality of ACPL
Encouraged by our small molecule results, we tested ACPL fur-
ther with recombinant proteins. Ubiquitin (Ub) is natively devoid
of cysteine27. We chose it as a model protein for our demonstration.
We produced recombinant native Ub and Ub with both a G76C
mutation and a C-terminal 6×His tag (Ub-G76C-6H) in E. coli and
purified them to homogeneity. We then ligated Ub-G76C-6H with
12 small molecule amines including Pa, allylamine (Aa), hydrazine
(Ha), and L- and D-amino acids (Figure 2A) by adding 5 mM
NTCB and a 50-1000 mM amine simultaneously to a 2 mg/mL Ub-
G76C-6H solution at pH 9 for an overnight incubation at 37 °C.
We carefully selected seven L-amino acids for our reactions to rep-
resent amino acids in different chemical categories and also differ-
ent sizes. For all tested compounds including proline that has a sec-
ondary amine and two D-amino acids, we obtained ligation prod-
ucts with 50-90% yields that were estimated by the SDS-PAGE
analysis of reaction mixtures (Figure S2, Table S2). After using
Ni2+ charged resins to simply remove unreacted intermediates, we
analyzed all 12 ligation products and the two original Ub and Ub-
G76C-6H proteins by electrospray ionization mass spectrometry
(ESI-MS) analysis. For all analyzed proteins, their deconvoluted
ESI-MS spectra displayed clearly observable monoisotopic peaks.
Since there is no commercial software for calculating protein mo-
noisotopic peaks, we wrote a Python script to calculate all theoret-
ical monoisotopic masses and their relative intensities for all pro-
teins and compared them to the determined ESI-MS spectra. Our
results showed that determined monoisotopic masses for all pro-
teins agreed very well with their theoretic values in terms of both
molecular weight and intensity (Figures S3-S16). Hydrolysis prod-
ucts were either non-detectable or at very low levels. To simplify
the comparison, we wrote another Python script to integrate decon-
voluted monoisotopic peaks and then calculate the average molec-
ular weights and intensities for all detected protein species in a par-
ticular spectrum. The final results are presented in Figures 2B and
2C. For all determined average molecular weights, they matched
their theoretical values with a deviation of ± 0.3 Da (Table S2). For
all 12 ligation products, we detected very few minor peaks in their
ESI-MS spectra indicating that all reactions were very selective.
One ligation product Ub-G76G is native Ub itself. Its ESI-MS
spectrum in Figure 2C matched that of recombinantly expressed
native Ub in Figure 2B. So far, our data demonstrated that ACPL
works exactly according to what we proposed on a recombinant
protein and this reaction is effective for amines that are primary,
secondary, Ha, and amino acids with different configurations, char-
acteristics, and sizes. The ligation with Ha was done in both native
and denatured conditions. The results from two conditions showed
minimal differences (Figures S16 and S17). Since conditions used
for ACPL so far were at 37 °C with a long incubation time that
might be a concern for some proteins, we also carried out reactions
under lower temperatures and shorter times. We performed the re-
action between Ub-G76C-6H and Pa under three different temper-
atures (8, 23, and 37 °C) as well as four different reaction times (4,
8, 12, and 16 h). The SDS-PAGE analysis of the final reaction mix-
tures exhibited no clear differences among all conditions. All these
conditions yielded the desired product as 50% (Figure S2E). There-
fore, a reaction at 8 °C for 4 h is well enough for ACPL.
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Figure 1. Protein synthesis by ligation techniques. (A) Native
chemical ligation and a derivative technique, peptide hydrazide li-
gation; (B) A proposed ACPL technique based on nucleophilic acyl
substitution of an activated cysteine residue in a recombinant pro-
tein by a nucleophilic amine. Without a nucleophilic amine, the
protein undergoes hydrolysis. When the nucleophile is hydrazine,
the afforded protein hydrazide can then undergo peptide hydrazide
ligation to form a larger protein.
Feasibility of ACPL
To demonstrate the feasibility of ACPL, we synthesized Boc-
Xxx-Cys-OMe dipeptides in which the Xxx identity varied be-
tween seven native amino acids including proline and carried out
their reactions with an equivalent amount of NTCB and then liga-
tion with propargylamine (Pa) in dichloromethane (DCM). Our re-
sult showed that all dipeptides reacted with Pa to form desired
products with varied yields (Table S1). Since the reactions were
performed in DCM, amino acids with a hydrophobic and large side
chain tended to have high yields, while amino acids with a rela-
tively small and hydrophilic side chain resulted in relatively low
yields. To further characterize the effect of the reaction on the chi-
rality of the amino acid at the N-terminal side of cysteine, we syn-
thesized the Boc-L-isoleucine-Pa using ACPL and compared it
with both Boc-L-isoleucine-Pa and Boc-DL-isoleucine-Pa that
Ubiquitin natively has a G75 residue that has the lowest steric
hindrance among all amino acids. In Ub-G76C-6H, the glycine im-
mediately N-terminal to G76C might have permitted easy pro-
cessing of the ligation. Other residues that have different chemical
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Figure 2. The synthesis of Ub conjugates by ACPL. (A) A schematic diagram to show the activation of recombinant Ub proteins containing
a cysteine by NTCB followed by nucleophilic acyl substitution with amines, both primary and secondary, to generate different Ub conjugates.
The native Ub has 76 residues and glycine at the 75th and 76th positions. (B) The deconvoluted and integrated ESI-MS spectra of wild type
Ub and Ub-G76C-6H. 6H represents a 6×His tag. (C) The deconvoluted and integrated ESI-MS spectra of Ub conjugates that were converted
from Ub-G76C-6H and had different ligated molecules at the G76 position. Pa, Ha, and Aa are three small molecule amines shown in A. All
other ligated molecules are amino acids whose one letter codes are used for labeling. All amino acids are in the L-configuration except two
D-amino acids with a footnote d. (D-E) The deconvoluted and integrated ESI-MS spectra of 7 recombinant Ub proteins and products of their
reactions with NTCB and Ha. C in Ub-C-6H represents cysteine. All detected molecular weights agreed well with theoretic values in a
deviation range of ± 0.3 Da.
properties and/or are sterically hindered might impede the ligation.
To resolve this concern, we mutated G75 in Ub-G76C-6H to six
other residues that are large in size, charged, and/or having a sec-
ondary amine, recombinantly expressed them, analyzed them with
ESI-MS (Figures 2D and S18-S23), and then reacted them in a one-
pot fashion with NTCB and Ha. We chose Ha in our demonstration
since its ligation products are protein hydrazides that can undergo
further peptide hydrazide ligation for making even larger proteins.
All reactions progressed exceedingly well and their reaction prod-
ucts displayed average molecular weights matching well to their
theoretic values (Figures 2E and S24-S29; Table S3), demonstrat-
ing that the residue immediately N-terminal to the targeted cysteine
has little detrimental effect on the ligation process. However, large
amino acids such as tryptophan did lead to lower yields in compar-
ison to small amino acids such as threonine (Figure S2, Table S3).
Putting a cysteine residue right after Ub G76 led to similar ligation
results with Ha, Aa, Pa and glycine with an at least 50 % conversion
rate (Figures 2D, 2E, and S30-S34 and Table S3). Ub has a flexible
C-terminus that may facilitate the ligation. To show that the liga-
tion may work in a more structurally constrained environment, we
introduced a cysteine mutation at K48 and K63, two residues in the
globular region of Ub and used the two afforded Ub mutants (Fig-
ures S35-S36 and Table S3) to undergo ACPL with Ha. ESI-MS of
reaction mixtures showed successful formation of two desired pro-
tein hydrazides (Figures S37-S38 and Table S3) indicating that
ACPL works well in a structurally constrained protein region. Li-
gation both in a structurally constrained protein region and under a
denatured condition is something that the traditional intein and lig-
ase-based methods cannot perform well. Collectively our data
strongly demonstrates the versatility of the ACPL technique.
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Figure 3. The synthesis of FLAG-Ub/Ubl-Pa and Ub/FLAG-SUMO1-3-AMC probes by ACPL and their applications in covalent
conjugation or activity assays of DUB/ULPs. (A) The deconvoluted and integrated ESI-MS of FLAG-Ub/Ubl-Pa and Ub/FLAG-SUMO1-
3-AMC probes. Ub-AMC was synthesized from Ub-G76C-6H. All other Pa- and AMC-conjugated Ub/Ubls were generated from FLAG-
tagged proteins. Ub/Ubls with their C-terminal glycine mutated to cysteine were expressed and purified as a protein fused with a N-terminal
FLAG tag and a C-terminal 6´His tag. ISG15, SUMO1-4, and MNSF have a native cysteine residue. This cysteine was mutated to alanine
or serine in all six expressed proteins for avoiding side reactions. The label “′” indicates this mutation. All detected molecular weights agreed
well with their theoretic values with a deviation range of 0.5 Da. (B) The formation of covalent adducts between FLAG-Ub-G76Pa and a
number of DUBs. Red arrows point to the generated adducts. (C) The formation of covalent adducts, indicated by red arrows, between
different FLAG-Ubl-GxPa probes and DUB/ULPs. (D) The DUB/ULP-catalyzed AMC release from Ub-AMC and three FLAG-SUMO-
AMC conjugates.
The Use of ACPL to synthesize Ub and Ub-Like Protein (Ubl)
Probes
Pa-conjugated FLAG-Ub (FLAG-Ub-G76Pa) to react with seven
DUBs and observed efficient covalent adduct formation for all
tested enzymes in both SDS-PAGE analysis and Western blotting
(Figures 3B and S63). We also performed similar tests for seven
Pa-conjugated FLAG-Ubl probes and observed their covalent bind-
ing to a number of ULPs as shown in Figure 3C. Some ULPs such
as SENP1 have only been vaguely confirmed in previous work to
deconjugate corresponding Ubls such as SUMO436. All synthe-
sized FLAG-Ub/Ubl conjugates, of which six are synthesized for
the first time, are activity-based probes that can be potentially used
to profile DUB and ULP proteomes in different tissues or cells. As
a demonstration, we incubated the HEK293T cell lysate with
FLAG-Ub-G76Pa and then probed the FLAG-Ub-conjugated pro-
teins by an anti-FLAG antibody in Western blotting. The result
showed the formation of a number of higher molecular weight spe-
cies compared to the original FLAG-Ub-G76Pa, indicating conju-
gation with other proteins in the cell lysate (Figure S64). However,
a control reaction using FLAG-Ub-G76C-6H showed no covalent
conjugation with any other proteins in the HEK293T cell lysate.
In eukaryotic cells, Ub and Ubls can be posttranslationally at-
tached to proteins for their functional regulation28-30. It has been
shown that replacing the C-terminal glycine in Ub and Ubl proteins
including SUMO1-3, NEED8, and ISG15 with Pa using either the
intein based approach or total synthesis afforded excellent probes
to conjugate covalently to deubiquitinases (DUBs) or ubiquitin-like
proteases (ULPs) that catalytically remove Ub or Ubls from their
conjugated proteins in cells31-35. To recapitulate these results and
demonstrate the broad application scope of our ACPL technique in
the probe synthesis, we recombinantly expressed Ub and a number
of Ubl proteins including SUMO1-4, NEDD8, ISG15, GABARAP,
GABARAPL2, UFM1, URM1, and MNSFβ (FLAG-Ub/Ubl-GxC-
6H: x denotes the terminal glycine position) that all contained a C-
terminal Gly-to-Cys mutation and were also fused with a N-termi-
nal FLAG tag and a C-terminal 6×His tag, purified them to homo-
geneity, and then carried out their reactions with Pa in the presence
of NTCB to afford their Pa-conjugated products. ISG15, SUMO1-
4, and MNSFβ natively contain a cysteine residue. This cysteine
was mutated to alanine or serine to avoid non-targeted reaction at
its location. The yields of all 12 reactions varied between 25% and
80% as indicated by their SDS-PAGE analysis (Figure S2, Table
S4). ESI-MS analysis of all 12 products showed their successful
and efficient synthesis (Figures 3A and S39-S62; Table S4). The
final results are presented in Figures 3A that displayed very little
side products for all 12 Pa-conjugated products. In comparison to
both intein based and total synthesis approach, our method for the
synthesis of these Pa conjugates is much simpler and easier to con-
trol. To reproduce some literature results, we used our synthesized
Ub and Ubls conjugated directly to 7-amido-4-methylcoumarin
(AMC) at their C-terminus are useful fluorogenic substrates of
DUBs and ULPs37. To demonstrate the synthesis of Ub/Ubl-AMC
conjugates using our ACPL technique, we made Ub-AMC and
FLAG-SUMO1-3-AMC by reacting recombinantly produced Ub-
G76C-6H and FLAG-SUMO1-3-GxC-6H proteins with Gly-AMC
in the presence of NTCB. The ESI-MS analysis of all four products
confirmed their successful formation (Figures 3A and S65-S68)
and the following activity assays showed that they served as active
substrates for cysteine proteases UCHL1 and SENP1, respectively
(Figure 3D). Overall, our combined data of Ub/Ubl probe synthesis
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establish the broad application scope of the ACPL technique and
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this technique can make Ub/Ubl probes readily available in a
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Figure 4. The synthesis of H2AK129ac and RNase H by ACPL (A) The deconvoluted and integrated ESI-MS spectra of H2A-K129C-
6H and H2AK129ac. H2A-K129C-6H was recombinantly expressed and then reacted with NTCB and Ne-acetyl-lysine to afford H2AK129ac.
(B) The synthesis of H2AK129ac, its isolation, and folding into an H2AK129ac/H2B dimer and then a nucleosome. The purification of
H2AK129ac was achieved by extracting the unreacted intermediate using Ni charged resins. (C) The deconvoluted and integrated ESI-MS
spectra of RNH59-196-K190C-6H, RNH59-189-Ha, and RNH59-196-K190C. RNH59-196-K190C-6H was recombinantly expressed in E. coli. It was
reacted with NTCB and Ha to afford RNH59-189-Ha that then underwent peptide hydrazide ligation with a 7-mer NH2-CADYGRK-OH peptide
to form a catalytic active RNH59-196-K190C. (D) The catalytic hydrolysis of an RNA substrate by RNH59-196-K190C. The RNA substrate had
a sequence 5’-Cy3-GACACCUGAUUC-Cy5-3’. A DNA fragment 5’-GAATCAGGTGTC-3’ was used to form a double strand with the
RNA substrate for binding to RNH59-196-K190C. The hydrolysis led to improved Cy3 (I3) and decrease Cy5 (I5) emission.
manner that can be performed in almost any biology lab for ad-
vancing Ub and Ubl biology studies.
H2AK129ac into a dimer with H2B and subsequently into a nucle-
osome (Figure 4B) making it possible to study effects of
H2AK129ac on the nucleosome structure and function. In the ESI-
MS spectrum of H2AK129ac in Figure 4A, we noticed a minor
peak at 13813.6 Da. H2A has two lysine residues, K125 and K127
that are adjacent to K129. Both crystal and cryo-EM structures
have indicated all three lysines are located at a flexible C-terminal
region of H2A39-40. Potentially either K125 or K127 can undergo
intramolecular cyclization with an activated K129C. These intra-
molecular cyclization products will have a theoretical molecular
weight (13813.9 Da) that matches the minor peak in the ESI-MS
spectrum. Based on the determined intensities of peaks in the ESI-
MS spectrum, this minor peak was lower than 15% of the desired
product demonstrating the selectivity of ACPL. By tuning the re-
action conditions, this minor peak might be further reduced.
The Use of ACPL to Synthesize H2AK129ac
To further demonstrate its broad application scope, we also car-
ried out ACPL to make non-ubiquitin or non-ubiquitin-like pro-
teins. In human cells, histone H2A can undergo posttranslational
acetylation at its terminal lysine K12938. The functional investiga-
tion of this acetylation such as how it influences the structure and
dynamics of the nucleosome will require the synthesis of the cor-
responding acetyl-histone, H2AK129ac. We chose to synthesize
H2AK129ac to demonstrate that our method can be applied to the
synthesis of histones with C-terminal modifications. As a matter of
fact, except H3 that natively has a cysteine, all other core histones
are lack of cysteine residues. We first recombinantly produced
H2A-K129C-6H, an H2A protein with a K129C mutation and a C-
terminal 6×His tag and then ligated it to Ne-acetyl-lysine with the
assistance of NTCB. The ESI-MS spectrum of the reaction product
showed the formation of H2AK129ac (Figures 4A and S69-S70;
Table S5) with an at least 60% yield. We folded successfully
The Use of ACPL in Combination with Peptide Hydrazide Li-
gation to Synthesize an Active RNase H
For all ligation reactions that we performed thus far, they in-
volved small molecules with only one amino group for avoiding
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side product formation. For ligation with larger molecules or pep-
ASSOCIATED CONTENT
Corresponding Author
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tides that have more than one amino group, one can couple ACPL
with peptide hydrazide ligation to resolve non-specificity issues.
To demonstrate this prospect, we recombinantly produced a B.
halodurans RNase H region with a C-terminal Cys-6×His tag
(RNH59-196-K190C-6H). Its ligation with Ha in the presence of
NTCB led to the synthesis of RNH59-189-Ha, a protein hydrazide
that we proceeded further to undergo peptide hydrazide ligation
with a 7-mer peptide, NH2-CADYGRK-OH to afford a ligated
product RNH59-196-K190C41. ESI-MS analysis showed the success-
ful synthesis of both RNH59-189-Ha and RNH59-196-K190C (Figures
4C and S71-S73; Table S5). Similar to what has been found in pre-
vious peptide hydrazide ligation reactions, we also detected a mi-
nor hydrolysis product at 15074.6 Da42. The ligated product
RNH59-196-K190C was catalytically active to hydrolyze an RNA
substrate as shown in Figure 4D. In the contrary, RNH59-189-Ha was
completely inactive toward this substrate. Our data related to the
synthesis of RNase H demonstrated that ACPL can couple to pep-
tide hydrazide ligation for conjugation with large peptides or even
protein fragments.
Correspondence and requests for materials should be addressed to
W.R.L. (wliu@chem.tamu.edu).
Notes
The authors declare no competing financial interests. All data sup-
porting the findings in this article are available in the main text or
the supplementary information. The two Python scripts are availa-
ble upon request.
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ACKNOWLEDGMENT
We thank Dr. Yohannes H. Rezenom who is the director of Texas
A&M University Chemistry Mass Spectrometry Facility for help-
ing us on the mass spectrometry analysis. This work was supported
by National Institutes of Health (grants R01GM127575 and
R01GM121584) and Welch Foundation (A-1715).
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In summary, we have developed a novel expressed protein liga-
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