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Biotechnology based process for production of a disulfide-bridged peptide Animesh Goswami, Steven L. Goldberg, Ronald L. Hanson, Robert M. Johnston, Olav K. Lyngberg, Yeung Y. Chan, Ehrlic T Lo, Steven H. Chan, Nuria de Mas, Antonio Ramirez, Richard Doyle, Wei Ding, Mian Gao, Stanley R. Krystek, Changhong Wan, Yeoun jin Kim, Deepa Calambur, Mark Witmer, and James W. Bryson Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00101 • Publication Date (Web): 20 Apr 2016 Downloaded from http://pubs.acs.org on April 21, 2016

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Bioconjugate Chemistry

Biotechnology based process for production of a disulfide-bridged peptide Animesh Goswami1*, Steven L. Goldberg1, Ronald L. Hanson1, Robert M. Johnston1, Olav K. Lyngberg1, Yeung Chan1, Ehrlic Lo1, Steven H. Chan1, Nuria de Mas1, Antonio Ramirez1, Richard Doyle2, Wei Ding3, Mian Gao4, Stanley Krystek4, Changhong Wan4, Yeoun jin Kim4, Deepa Calambur4, Mark Witmer4, James W. Bryson4* 1

Chemical Development, R&D, Bristol-Myers Squibb, 1 Squibb Drive, New Brunswick, NJ 08903,

2

Chemical Development Operations, R&D, Bristol-Myers Squibb, New Brunswick, NJ 08903,

3

Analytical and Bioanalytical Development, R&D, Bristol-Myers Squibb, New Brunswick, NJ 08903,

4

Discovery, R&D, Bristol-Myers Squibb, Route 206 and Province Line Road, Princeton, NJ 08543,

* Corresponding author: Animesh Goswami, Chemical Development, R&D, Bristol-Myers Squibb, 1 Squibb Drive, New Brunswick, NJ 08903, USA, E-mail: [email protected], Tel: 732-227-6225, FAX: 732-227-3994.

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ABSTRACT A disulfide-bridged peptide drug development candidate contained two oligopeptide chains with eleven and twelve natural amino acids joined by a disulfide bond at the N-terminal end. An efficient biotechnology based process for the production of the disulfide-bridged peptide was developed. Initially, the two individual oligopeptide chains were prepared separately by designing different fusion proteins and expressing them in recombinant E. coli. Enzymatic or chemical cleavage of the two fusion proteins provided the two individual oligopeptide chains which could be conjugated via disulfide bond by conventional chemical reaction to the disulfide-bridged peptide. A novel heterodimeric system to bring the two oligopeptide chains closer and induce disulfide bond formation was designed by taking advantage of the self-assembly of a leucine zipper system. The heterodimeric approach involved designing of fusion proteins with the acidic and basic components of the leucine zipper, additional amino acids to optimize interaction between the individual chains, specific cleavage sites, specific tag to ensure separation, and two individual oligopeptide chains. Computer modeling was used to identify the nature and number of amino acid residue to be inserted between the leucine zipper and oligopeptides for optimum interaction. Cloning and expression in rec E. coli, fermentation, followed by cell disruption resulted in the formation of heterodimeric protein with the inter-chain disulfide bond. Separation of the desired heterodimeric protein, followed by specific cleavage at methionine by cyanogen bromide provided the disulfide-bridged peptide. INTRODUCTION The disulfide-bridged peptide (Figure 1) is a delta protein kinase C inhibitor under development for the treatment of injury associated with ischemia and reperfusion of acute myocardial infarction.1-3 The disulfide-bridged peptide is composed of a protein kinase C (referred henceforth as “PKC” or “cargo”)


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active portion joined to a transporter (referred henceforth as “TAT” or “carrier”) fragment by a disulfide bond between the two N-terminal cysteines of the two peptide chains composed of all natural amino acids. The active PKC inhibitory fragment is an undecapeptide. The TAT fragment facilitating cellular uptake is a dodecapeptide rich in basic amino acids.

Figure 1: Disulfide-bridged peptide PKC, Cargo

CSFNSYELGSL CYGRKKRRQRRR TAT, Carrier

The peptide has been synthesized by conventional solid phase peptide synthesis of the PKC and TAT components followed by chemical conjugation. Though solid-phase peptide syntheses have been used for the synthesis of peptides up to about 50 amino acids, they suffer from a number of disadvantages, e.g. repeated protection-deprotection, low overall yield, low productivity, a large number of impurities generated and carried over in successive steps requiring extensive purification, consumption of large amount of solvents and generation of significant waste materials. A chemical ligation method is used for chemical synthesis of larger peptides of up to about 200 amino acids.4 Biotechnology (recombinant DNA and fermentation) is generally used for the synthesis of much larger proteins. This paper describes an efficient alternative biotechnology based method for the production of the disulfide-bridged peptide.



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RESULTS AND DISCUSSION The peptide is composed of all natural amino acids thus allowing the conventional recombinant DNA technologies for the synthesis. However PKC and TAT fragments are joined by a disulfide bond, and therefore a ribosome mediated direct construction of the entire disulfide-bridged peptide in one contiguous peptide chain is not feasible. Our first approach was construction of two (PKC and TAT) individual chains separately, followed by joining the two fragments by disulfide bonds by conventional chemical methods. Preparation of individual chains (PKC and TAT) Design of fusion proteins PKC and TAT fragments have only 11 and 12 amino acids, respectively. Such small peptides are expected to be proteolytically degraded by microorganisms and are unlikely to be produced in any meaningful quantity via fermentation. Indeed, complete degradation of the disulfide-bridged peptide was seen when it was added to growing E. coli cells. A general methodology to circumvent such an issue is to attach the peptide to a protein partner to generate a fusion protein with an affinity tag for the recovery and separation of the desired protein.5-11 The desired PKC or TAT fragments were prepared using such fusion proteins followed by specific cleavage by either chemical or enzymatic processes. The basic model of the fusion protein is shown in Figure 2.


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Figure 2: Fusion protein for production of PKC or TAT

Fusion Partner

Affinity Purification

Cleavage Site

PKC or TAT

a.

MBP

His6

DDDDK

PKC or TAT

b.

IF2

His6

M or W

PKC or TAT

c.

SET

His6

M

PKC or TAT

d

SET

His6

DDDDK

PKC or TAT

e

SUMOInv

His6

GG

PKC or TAT

f

DnaB/gyrA

CBD

Not applicable

PKC or TAT

Maltose binding protein (MBP) The first fusion protein was designed with maltose binding protein (MBP) as fusion protein partner, histidine tag for affinity purification and a cleavage site for enterokinase (EK) cleavage (Figure 2a). The maltose binding protein (MBP) was chosen for good expression in E. coli and to promote solubility of the fusion protein.12, 13 The affinity separation via maltose binding is not very selective. A polyhistidine segment was reported to be extremely selective and efficient for purification by affinity chromatography over metal (especially nickel) chelated adsorbent.14-17 An enterokinase cleavage ACS Paragon Plus Environment


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recognition sequence (DDDDK-X) was absent in both maltose binding protein and in the desired target proteins PKC or TAT. Furthermore, enterokinase is reported to be quite specific and is not influenced by the downstream amino acid residues.18-20 Synthetic genes encoding MBP-His6-EK-PKC and MBP-His6-EK-TAT were cloned using our proprietary vector pBMS200421 and expressed in E. coli. The initial fusion proteins contained 2.79% TAT and 3.8% PKC by mass. After cleavage by enterokinase, HPLC and LC-MS confirmed that the PKC and TAT samples derived from this process matched the retention time, mass and sequences for the authentic PKC and TAT peptides (see Supporting Information SI). The yields of PKC and TAT were found to be 1.61% and 1.33%, respectively, from the fusion proteins. Expression, affinity purification and cleavage of this fusion protein worked well but the yield of the peptide products was limited by their small size relative to the size of the fusion proteins. The maltose binding protein is rather large (MW ~40KD). The maximum theoretical weight % yield of PKC or TAT from the fusion protein is only about 4% making it a very inefficient process. The molecular weight of the fusion partner should ideally be as low as possible to provide highest weight % yield of PKC or TAT. Enterokinase is very selective, but relatively expensive enzyme. A selective and inexpensive cleavage reagent is desirable. Chemical cleavage of proteins at methionine by inexpensive cyanogen bromide (CNBr) is selective.22 Though CNBr is toxic and has to be handled under appropriate conditions, it has been used successfully for insulin production.23 Initiation factor 2 (IF2) Ideally, a fusion partner should fulfill two main requirements. It should be well-expressed in soluble form in the heterologous host strain and relatively small in size so its mass relative to the target protein is minimized. These requirements are met by domain I of initiation factor 2 (IF2) of E. coli, which is required for translation of proteins. Sorenesen et al described its use in producing soluble streptavidin.24 Domain I consists of 158 residues of the N-terminus of IF2 (Figure 3). Another advantage of this fusion


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partner is the absence of an internal methionine or tryptophan residue, which can be located immediately prior to the target peptide for possible chemical cleavage. Figure 3: Domain I of IF2 MTDVTIKTLAAERQTSVERLVQQFADAGIRKSADDSVSAQEKQTLIDHLNQKNSGPDKLTLQR KTRSTLNIPGTGGKSKSVQIEVRKKRTFVKRDPQEAERLAAEEQAQREAEEQARREAEESAKRE AQQKAEREAAEQAKREAAEQAKREAAEKDKVS

Synthetic genes encoding IF2-His6-Met/Trp-PKC and IF2-His6-Met/Trp-TAT) containing methionine or tryptophan as the cleavage site were created (Figure 2b) and all four of them were expressed in E. coli. IF2 fusion proteins with methionine cleavage sites were obtained in good yield in soluble form and provided ~13% yield of PKC or TAT after CNBr cleavage. IF2 fusion proteins with tryptophan cleavage sites were also prepared. Cleavage with 2-(2-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine (BNPS-skatole)25 provided the PKC peptide. But, it was not possible to unambiguously establish the formation of TAT peptide by cleavage with BNPS-skatole. Solubility enhancement tag (SET) The amino acid sequences of the solubility enhancement tag (SET) series of leader sequences are part of the VariFlex pBE vectors. These confer a negative charge to the fusion protein, which is speculated to play a role in their ability to enhance solubility to partners otherwise insoluble when expressed in E. coli.26 Particularly attractive is their very small size, in this instance representing only 80% of the total mass vs. 96% for fusions with maltose binding protein equivalent to a 5-fold increase in the amount of desired PKC or TAT peptide per molecule. There is no methionine or tryptophan in the amino acid sequence of SET protein that may undergo chemical cleavage. The enterokinase cleavage sequence



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(DDDDK) is also absent in SET thereby allowing the cleavage by enterokinase of fusion protein using SET as a partner. SET fusion proteins (SET-His6-M-PKC and SET-His6-M-TAT) with methionine cleavage site (Figure 2c) were expressed in E. coli. Cleavage of fusion protein SET-His6-M-PKC by CNBr provided PKC. However, no TAT was detected from the cleavage of SET-His6-M-TAT by CNBr. Instead, a truncated TAT peptide with loss of two terminal arginine was observed. Though enterokinase cleaved both SET fusion proteins (SET-His6-EK-PKC and SET-His6-EK-TAT) containing the enterokinase cleavage site (Figure 2d), intact PKC or TAT could not be detected. Individual chain approach – Summary of achievements and its inefficiency for a suitable process The fusion protein approach demonstrated the formation of PKC and TAT fusion proteins with different partners, e.g. MBP, IF2, and SET proteins. Cleavages of fusion proteins at specific sites were achieved providing PKC and TAT. Enterokinase was found to be an effective enzyme for cleavage of MBP fusion proteins. Cyanogen bromide was found to be the best chemical cleavage reagent providing PKC and TAT with IF2 fusion proteins and PKC with SET fusion protein. BNPS-skatole cleavage was successful to provide PKC with IF2 fusion protein. The PKC and TAT peptides produced by individual chain approaches were of very high purity and were identical with those obtained from conventional solid phase peptide synthesis. With low molecular weight fusion protein partners, e.g. IF2 and SET, it was possible to produce both PKC and TAT in higher yields. Attempts to prepare the individual chains by using SUMO and Intein as fusion proteins (see SI) were not successful. In spite of the success of individual chain approaches, potential issues remained to develop an efficient process for the production of PKC and TAT and for the disulfide-bridged peptide. PKC and TAT proteins are expressed in two rec E. coli strains requiring two fermentations, two cell processings to make the protein solutions, two affinity chromatographies to separate the respective fusion proteins, and two cleavages followed by isolation to get the PKC and TAT peptides. The conjugation of PKC and TAT


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peptides to make the complete disulfide-bridged peptide has to be carried out by conventional chemical processes and thereby suffers from the yield loss due to statistical distribution (homo and hetero conjugations) and impurities of that process. A biotechnology based process where the entire disulfide-bridged peptide molecule with the disulfide bond between PKC and TAT can be formed in one recombinant system by a single fermentation followed by single successive isolation and separation steps is desirable for an efficient process. Preparation of intact disulfide-bridged peptide Design of heterodimeric fusion proteins The leucine zipper motif leads to the formation of heterodimeric proteins, e.g., Jun and Fos oncoproteins where the two protein chains are brought closer together and form a coiled coil structure by electrostatic interactions between the basic Jun and acidic Fos proteins.27, 28 The preferential formation of heterodimer over homodimers and increased stability of heterodimeric coiled coil can be achieved by appropriately designing the Jun and Fos components (Moll et al, 2001; Mason and Arndt, 2004; Mason et al, 2006).29-31 The self-assembling capability of the acidic and basic components of leucine zipper system was utilized to bring the two oligopeptide chains closer and induce disulfide bond formation. Our design strategy was started from one of the best heterodimerizing Jun-Fos combination28 (Figure 4) referred henceforth as RR and EE, respectively. Figure 4: Jun (RR) and Fos (EE) sequences:

RR: KGGGLEIRAAFLRRRNTALRTRVAELRQRVQRARNRVSQYRTRYGPL EE: SSGLEIEAAFLEQENTALETEVAELEQEVQRLENEVSQYETRYGPLGGGK



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The cargo PKC was attached to the C-terminal end of the acidic EE portion and the carrier TAT was attached to the C-terminal end of the basic amino acid rich RR using a methionine at the joining sites for future cleavage. Methionine was specifically selected for the cleavage site after considering multiple factors, e.g. simple and low cost chemical cleavage by CNBr and minimum perturbation of coiled coil structure by one amino acid. The hypothesis was that after the RR-TAT and EE-PKC were expressed, the heterodimer would be formed which would bring the two chains closer together to induce disulfide bond formation between the PKC and TAT. Once the disulfide bond is formed, CNBr cleavage at the methionine would liberate disulfide-bridged peptide molecule with the PKC-TAT joined together by a disulfide bond. In order to achieve separation, His6 was added on the N-terminal end of EE-PKC. We also added a Myc tag on the N-terminal of the RR-TAT component to facilitate detection during the development work. Though Myc based affinity separation can be used, His6 was used in the production process due to its simplicity and lower cost. Before the construct was created, computer simulation of the RR-M-TAT and EE-M-PKC components suggested some gaps between the cysteine residues in the two protein chains which might not be optimum for a disulfide bond formation. Molecular modeling suggested that addition of one to three alanine before the methionine in the basic chain (RR) and zero to one alanine in the acidic (EE) chain would bring the two chains in the optimum position for the formation of disulfide bond while maintaining the strong interaction in the coiled coil structure (Figure 5). The basic design and conceptual process flow chart is shown in Figure 6.


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Figure 5: Model of heterodimer focusing on disulfide region



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Figure 6. Basic design and conceptual process chart of leucine zipper expression

Generation of initial constructs and selection of the best construct for development Six bicistronic constructs were created with varying numbers of alanines in different chains as suggested by computer modeling (Table 1). All six proteins were expressed in E. coli BL21(DE3) and Rosetta (DE3) strains which were positive to both anti-His and anti-Myc antibodies suggesting that dimers contained both proteins.


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Table 1. Bicistronic constructs with varying alanine spacer

Disulfide-bridg ed peptide produced after ID

Plasmid

CNBr cleavage (mg per L of flask culture)

A

Myc-RR-A-M-TAT-His-EE-M-PKC-pET28

B

Myc-RR-AA- M-TAT-His-EE-M-PKC-pET28

34

C

Myc-RR-AAA-M-TAT-His-EE-M-PKC-pET28

32

D

Myc-RR-A-M-TAT-His-EE-A-M-PKC-pET28

27.5

E

Myc-RR-AA-M-TAT-His-EE-A-M-PKC-pET28

15

F

Myc-RR-AAA-M-TAT-His-EE-A-M-PKC-pET28

14

28.5

By visual inspection of SDS gels it appeared that the BL21 strains had more of the hetero fusion construct. Also by visual inspection the construct B (Myc-RR-AA-M-TAT-His-EE-M-PKC) was the most abundant of the three BL21 samples, while the Rosetta samples looked virtually identical. The soluble cell lysates from all were purified on Ni-NTA columns by utilizing His-tag affinity. LC-MS confirmed the formation of heterodimer. For example, construct B showed an intact protein with 16733 Dalton as expected for Myc-RR-AA-M-TAT + des-met-His-EE-M-PKC. Reduction by dithiothreitol



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(DTT) showed two components 8804 Da (Myc-RR-AA-M-TAT) (confirmed sequence MEQKL), and 7930 Da des-met-His-EE-M-PKC (confirmed sequence GSSHH) (Figure 7). Figure 7. LC and LC-MS of construct B after reduction by DTT.

a

b

c

a. LC of construct B + DTT, b. LC-MS of peak at 4.56 min TAT fusion protein, c. LC-MS of peak at 6.27 min PKC fusion protein

Circular dichroism analysis of the heterodimeric proteins showed folded and thermally stable structure even in 6M urea (See SI). The heterodimeric proteins from all six constructs were cleaved by CNBr and the formation of disulfide-bridged peptide was confirmed by HPLC comparison with authentic sample and LC-MS. The amounts of disulfide-bridged peptide produced were quantified by HPLC and the results showed that construct B produced the highest amount. The construct B (Figure 8) with two alanines before the TAT and no alanine before PKC was the best and selected for further development.

Figure 8. Best construct of heterodimeric leucine zipper fusion protein for production of disulfide-bridged peptide ACS Paragon Plus Environment

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His-EE-M-PKC 1 MGSSHHHHHH SSGLEIEAAF LEQENTALET EVAELEQEVQ RLENEVSQYE 51 TRYGPLGGGK MCSFNSYELG SL Myc-RR-AA-M-TAT 1 MEQKLISEED LKGGGLEIRA AFLRRRNTAL RTRVAELRQR VQRARNRVSQ 51 YRTRYGPLAA MCYGRKKRRQ RRR

Process development for production of intact disulfide-bridged peptide Cloning and Expression: In addition to the above construct B with pET, our proprietary vector pBMS2004 was chosen for process development work. The best construct B (Myc-RR-AA-M-TAT-His-EE-M-PKC) gene sequence was amplified, ligated to plasmid vector pBMS2004, and then introduced into several E. coli strains which had proven to be good for expression of heterologous proteins based on our previous experience or were reported to be capable of expression of toxic proteins (BL21, JM110, W3110-M25, C41 and C43). Flask expression studies were performed to determine the optimum growth temperature and IPTG concentrations for various strains containing the plasmids. Expression levels using the proprietary vector were improved. All proteins were soluble and very good expression levels were achieved. Although the greatest amount of protein was obtained in strain C43, it was noted that these high expression levels correlated inversely with growth, suggesting that expression of the heterodimeric proteins was somewhat toxic to E. coli. The unusually basic nature of the TAT portion may in particular be responsible for this response to induction. This would in turn suggest that induction at larger scale should be performed once ACS Paragon Plus Environment


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the cells have reached a moderate to high density. Fermentation: Initial small scale fermentations (5L scale, see SI) showed promising results with W3110-M25, BL21 Gold and BL21(DE3) systems. Optimization studies were performed examining media, addition of supplements, and time of induction and harvest. Plasmid vector pBMS2004 produced significantly more heterodimeric protein than pET vector. Induction of recombinant protein synthesis led to a noticeable decrease in the growth rate of the culture. Due to the apparent toxic effect of the expressed heterodimeric proteins on E. coli growth, growth of E. coli cells expressing the heterodimeric fusion proteins was greatly reduced and limited the accumulation of cell mass after induction. High protein productivity was achieved by IPTG induction at high cell density. A cell mass of 100 g/L with heterodimeric protein titer of 80mg/L was obtained by carrying out the entire fermentation at 37°C. The strain W3110-M25 showed the best results from 5 L to 15 L scale and was selected for further scale up. A strategy of building up as much cell mass as possible prior to induction was implemented in scale up to 250L scale fermentation. In addition, a literature report suggested that full induction of promoters in very dense cultures may require increasing the inducer concentration over that used under shake flask or batch fermentation conditions.32 While 0.5 mM IPTG was found to be optimal for fermentation in flask, a much higher amount of IPTG was found to be optimal for high density process in fermentors. The 250 L scale fermentations were stopped in about 4-5 hours post-induction when the heterodimeric protein production reached a maximum resulting in a cell density of about 60-80 g/L providing 7-9 mg of fusion protein per g of cells. Isolation of heterodimeric fusion protein In addition to the desired heterodimeric protein, the cell extracts contained varying levels of homodimers and truncated hetero- and homo-dimers. The fermentation condition is optimized to provide ACS Paragon Plus Environment

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maximum amounts of the desired intact heterodimer. Initially the protein in the eluent from the Ni Sepharose column was found to precipitate on standing. Addition of 2 M urea in all the buffers used for the preparation prevented the precipitation. Urea can interfere with the electrostatic interaction between the chains, and as expected, homodimer content is increased by increasing the urea concentration. A concentration of 2M urea is optimum to maximize solubility and minimize perturbation of heterodimeric interaction. Imidazole concentration in the column wash and elution buffers was optimized to remove as many impurity proteins as possible while retaining the fusion protein. Although truncated fusion protein and truncated PKC dimers were found in all preparations, incubation of fusion protein samples with cell extracts did not increase the truncation, nor did the inclusion of bacterial protease inhibitor cocktail in the binding/cell disruption buffer affect the ratio of intact/truncated proteins (see SI). Best yield of fusion protein heterodimer was obtained at pH 8 with 2M urea, 0.5 M NaCl and 0.5 M imidazole. The concentration of the intact fusion protein heterodimer remained stable in this solution for 13 days. Best recovery of fusion protein was obtained by processing the clarified cell extract immediately on the Ni sepharose column, but extracts could be held at 4oC for six days before the affinity column with only a small decrease in yield and little effect on the ratio of intact/truncated fusion protein or heterodimer/homodimers. One day of aging after the affinity column gave a maximum yield of the disulfide-linked heterodimer, whereas the extract held for 1 day was only about half converted to the disulfide-linked heterodimer. Formation of the disulfide-linked dimer occurred more quickly after the protein had been purified on the Ni sepharose column rather than in the extract, probably because of the removal of reducing agents found in the cell extract by the affinity purification. In initial experiments ethanol or trichloroacetic acid were used to precipitate the fusion protein from the column eluate prior to CNBr cleavage, but better results were obtained by using the eluate directly for the CNBr cleavage. In large scale operation, 11 kg E. coli cell paste provided 55.8 g intact heterodimeric fusion protein ACS Paragon Plus Environment


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with peak area ratios of intact/truncated fusion protein ranging from 4.47 to 4.60 and peak area ratios of hetero/homodimers ranging from 2.86 to 3.25. Cyanogen bromide cleavage of fusion protein and isolation of disulfide-bridged peptide: The efficiency of the chemical cleavage by CNBr depended on a complex balance between the relative rates of, at least, three general pathways: (a) the desired iminolactone hydrolysis, (b) an unproductive methionine to homoserine conversion (Figure 9), and (c) degradation of disulfide-bridged peptide under the reaction condition. Figure 9. CNBr cleavage of fusion protein

NH

S Me

O

NH

O

H N

NH

CNBr NC

R'

S Me

O

NH HBr H N OH

- NC SMe

R'

O

H N

O

Br

H2O

R' Br

NH

O

O

H N

O

O

O

NH

O

H H N

O

R' H

O

HBr H2N

Desired O

R'

Br

R' NH

O

O

H H N

O

HBr NH

Undesired

R' H Br

HO

O

O

H N R'

Kinetic and spectrometric investigations led to an improved cleavage condition and enabled an efficient process. The reaction was carried out using the fusion protein solution directly obtained from the Ni-purification column. Cleavage of fusion protein with 120 equiv of CNBr was completed after 17 hours


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at 25°C. Removal of toxic MeSCN and excess CNBr was accomplished by distillation into hypochlorite. The yield of the desired disulfide-bridged peptide was about 50% due to the undesired homoserine side reaction. Reversed phase chromatography of the reaction product followed by lyophilization of the eluted fraction provided disulfide-bridged peptide in about 75% yield and of excellent purity HPLC AP>99 (Figure 10). The identity of the disulfide-bridged peptide was confirmed by LC-MS (Figure 11) and other physical data (see SI) and by comparison with the material previously prepared by solid phase peptide synthesis. Furthermore, the disulfide-bridged peptide obtained from this process had significantly lower number and also lower amounts of impurities than that obtained via solid phase peptide synthesis. Figure 10. HPLC of the disulfide-bridged peptide obtained from heterodimeric fusion protein

300

300

Solvent Peaks

Disulfide-bridged peptide

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100

100

0 0

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Figure 11. LC-MS of the disulfide-bridged peptide obtained from (a) solid phase peptide synthesis, and (b) heterodimeric fusion protein

960.8 960.8 961.2

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960.5

960.9 961.2 960.6

961.9

0 960

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m/z 964

961.9

961.9

961.6

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961.9 720.9

1497.8

961.6

%

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960.9 961.2 100

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%

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962.2

1440.9 1497.9

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0 600

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1600

0 600

m/z 800

a

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a. The theoretical monoisotopic mass of the disulfide-bridged peptide is 2878.5 Da; while the measured monoisotopic mass calculated from the most abundant [M+3H]3+ ion at m/z 960.5 was 2878.5 Da, consistent with the expected mass of the peptide. No lockmass correction was applied. The ion at m/z 1497.8 was the TFA adduct ion to m/z 1440.8. The insert shows the isotope-resolved [M+3H]3+ ion in the expanded spectrum. b. The theoretical monoisotopic mass of the disulfide-bridged peptide is 2878.5 Da; while the measured monoisotopic mass calculated from the most abundant [M+3H]3+ ion at m/z 960.6 was 2878.8 Da, consistent with the expected mass of the peptide. No lockmass correction was applied. The ion at m/z 1497.9 was the TFA adduct ion to m/z 1440.9.

The leucine zipper based approach led to a novel method for producing the disulfide-bridged peptide in high overall yield, high productivity, excellent quality, requiring only one final chromatography, and is significantly “green” with minimum process waste.


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EXPERIMENTAL PROCEDURES HPLC of Peptides The disulfide-bridged peptide standard solution was prepared (1 mg/ml) in CH3CN-water (10:90) containing 0.1% TFA. For reduction of the disulfide-bridged peptide, tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) was added and the mixture was incubated overnight. Samples were analyzed with a Peeke Scientific Ultro 120 C18Q column (15x0.46 cm, 5 µΜ) at 25 °C with UV detection at 214 and 280 nm and an injection volume of 5 to 20 µl. Mobile phase A contained 0.05% TFA in water and B contained 0.05% TFA in CH3CN. The mobile phase was a gradient of 5 to 50% B from 0 to 25 min, 50% to 90% B to 28 min, 90% B to 34 min, ran at a flow rate of 1 mL/min. Retention times were: TAT 6.5 min, TAT homodimer 8.6 min; Disulfide-bridged peptide (the heterodimer TAT-PKC) 13.7 min, PKC 16.6 min. PKC homodimer18.6 min. HPLC of heterodimeric (leucine zipper LZ) fusion proteins (LZPKC or LZTAT) Method 1 Samples were analyzed with a YMC basic column (25x0.46 cm, 5µm) at 25 °C with UV detection at 215 and 280 nm. Mobile phase A contained 0.15% TFA in water-CH3CN 95:5and B contained 0.15% TFA in water-CH3CN 50:50. Samples of 0.1 ml typically were diluted with 0.2 ml 1N HCl + 0.7 ml mobile phase A, filtered, and 15 µl injected on the column. The amount of HCl added was increased for more concentrated samples to bring the protein into solution. The mobile phase was a gradient of 25 to 100% B from 0-30 min, 100% B from 30-40 min ran at a flow rate of 1 mL/min. and an injection volume of 15 µl. Retention times were LZPKCTAT 17.2 min, truncated LZPKCTAT 16.7 min, LZPKC homodimer 16.1 min, half truncated LZPKC/PKC homodimer 15.8 min. Concentration of fusion protein was calculated from the area of the fusion protein peak relative to the peak area of a bovine serum albumin



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(Pierce) standard at 280 nm correcting for the difference in extinction coefficients.. Method 2. Samples were analyzed with a Waters XBridge BEH300 C18 column (15x0.46 cm, 3.5µm) maintained at a temperature 45 °C and UV detection at 215 and 280 nm and a sample injection volume of 10 µl. Mobile phase A contained 0.05% TFA in water and B contained 0.05% TFA in CH3CN. The mobile phase was a gradient 16 to 50% B from 0-46 min, 50-95% B from 46-57.6 min ran at a flow rate of 1 mL/min. Retention times were: LZTAT monomer 17.0, LZTAT dimer 19.0 min; truncated (PKC 2-61/or 62) LZPKCTAT 31.5 min; LZPKCTAT 32.1 min; LZ half truncated PKC/PKC homodimer 33.4 min; LZPKC homodimer 34.1 min; LZ truncated PKC monomers 38.4 min; LZPKC monomer 38.6 min. The gradient was scaled accordingly for the UPLC column (10X0.21 cm, 1.7 µm). The LC-MS methods used high resolution and accurate mass (HRAM) spectrometry with Thermo Orbitrap Discovery or Waters Q-TOF Premier mass spectrometers. Protein analysis Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out with Invitrogen Nupage bistris 10 or 12% 1 mm 10 lane gels. MOPS or MES running buffer, non-reducing or reducing conditions, and SeeBlue®Plus2 marker proteins were used. Results were generally for qualitative purposes but quantitation of LZPKCTAT was done using a Carestream Health GelLogic 2200 Digital Analyzer with lysozyme as a standard. LZ PKCTAT samples were also analyzed with an Agilent 2100 Bioanalyzer using the Protein 80 kit using reduced or non-reduced samples. Quantitation is from an internal standard. Total protein was measured with the BioRad protein assay. Elution from the 10 ml Ni-Sepharose column was monitored at 280 nm. Cloning and expression of individual chain constructs


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Synthetic genes for fusion proteins (Figure 2) were prepared, cloned in plasmid pBMS2004 and expressed in E. coli BL21Gold cells. Cells were grown at 30°C, 250 rpm for 22 hr in MT5-M2 Km media (2% pea hydrolyzate, 1.85% yeast extract, 0.6% Na2HPO4, 0.125% (NH4)2SO4, 4.0% (w/v) glycerol, NaOH to pH 7.2, autoclave, sterile kanamycin sulfate 50 µg/mL) followed by IPTG induction. Harvested cells were suspended in phosphate buffer pH 7 containing1 mM DTT. Cells were broken and the soluble and insoluble portions were separated. SDS-PAGE analysis of the solution showed that both desired fusion proteins were highly overexpressed and the heterologous proteins were found almost exclusively in the soluble protein fraction. Various E. coli strains were used for expression, e.g. BL21Gold, BL21(DE3), MM294, W3110, LE392. IF2, SET, SUMO, and Intein fusion protein expressions with different cleavage sites were carried out in a similar manner by following standard procedures. (See SI for details) Isolation of Maltose binding fusion protein and generation of PKC and TAT Soluble fractions from rec E. coli cells (1.98 g TAT-fusion protein cells or 1.79 g PKC-fusion protein cells) were purified on 2mL Ni-NTA columns with buffers containing increasing imidazole concentration. The fractions containing the purified fusion proteins were identified using SDS gels and dialyzed into the enterokinase cleavage buffer (20 mM Tris-chloride pH 8, 50 mM NaCl, 2 mM CaCl2). To 1.7 ml samples containing 29 mg MBK-TAT or 43 mg MBP-PKC were added a 34 µl solution containing 0.068 µg enterokinase (New England Biolabs) (~0.0002% w/w of fusion protein) and the samples were incubated for 19h at 28oC for enterokinase cleavage. Samples were analyzed by SDS-PAGE, HPLC, and LCMS(See SI) . Creation and expression of heterodimeric fusion protein constructs All constructs (Table 1) were generated by subcloning the synthetic bi-cistronic inserts as a



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NdeI-XhoI fragment using a pET28 vector and confirmed by DNA sequencing. All six proteins were expressed in both E.coli BL21(DE3) and Rosetta(DE3). Growth at 20°C of the initial constructs in TB/autoinduction media facilitated soluble protein production. Cells were lysed and separated based on His-tag affinity on Ni-NTA columns as above for the individual chains. SDS-PAGE under non-reducing conditions and LC-MS analysis demonstrated the presence of disulfide bonded fusion proteins. The fusion proteins were cleaved by CNBr and the results are shown in Table 1. Heterodimeric fusion protein construct for process development The bicistronic insert present in B (Table I) (myc-RR-AA-M-carrier-His-EE-M-cargo-pET28, GenBank Accession number KU685481) was amplified for digestion and ligation into pBMS2004. Restriction digestions with NdeI and KpnI were performed. Single kanamycin-resistant colonies with confirmed pBMS2004-myc-RR-AA-M--carrier-His-EE-M-cargo plasmids were transformed by electroporation into different competent E. coli strains, e.g. BL21Gold, W3110-M25, JM110, C41, and C43. Ten mL of MT5-M2 Km medium in a 50 mL flask was inoculated with a single colony of each strain. Flasks were incubated at 37°C, 250 rpm. The culture was diluted into fresh MT5-M2 Km medium and grown at various temperatures between 2°C and 30°C and induced with varying concentration of IPTG (50 µM to 1 mM). After appropriate time, centrifugation (5,000 g) gave the cell pellets. Approximately 0.1 g of cells were resuspended in 1 mL imidazole lysis buffer (50 mM sodium phosphate - 10 mM imidazole - 300 mM NaCl pH 8) and disrupted to release the proteins. The total protein solution was centrifuged at 18000g for 5 min to separate the soluble fraction. Fermentations in 250 L scale for production of heterodimeric protein Fermentation was scaled up from flask to 5L (see SI) and finally to 250L scale. Two frozen vials each containing one mL of culture was thawed to room temperature. About 0.5 mL of the culture was used ACS Paragon Plus Environment

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to inoculate each of the four 4L flasks containing 1 L MT5-M2 Km medium. Flasks were shaken at 30°C, 250 rpm, for one day. The grown culture was added to 150 L of sterilized MT5-M2 Km medium. The culture was grown at 37°C with aeration. Induction with 4 mM to 20 mM of filter sterilized IPTG solution was done at about 15-18 hours at OD600 ~ 26 and cell density ~ 62 g/L. Fermentations were stopped in about 4-5 hours post-induction when the heterodimeric protein production reached a maximum with moderate increase in OD600 ~ 35-45 and cell density ~ 60-80 g/L providing 7-9 mg of fusion protein per g of cells. The cells were harvested by centrifugation, broken and proteins produced were analyzed as described before. Isolation of heterodimeric protein in large scale from the E. coli cell paste E. coli cell paste (11 Kg) was suspended at 15% w/v in binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 2 M urea, 20 mM imidazole pH 8) and disrupted by microfluidization at 12,000 -15,000 psig. Cell debris was removed by passage through a Sharples centrifuge and then clarified by tangential flow filtration (See SI for details). The recovery of the fusion protein in the filtration steps was 62 to 76%. Loading studies indicated up to 490 ml filtered extract could be applied to a 10 ml Ni sepharose column without breakthrough of the fusion protein. The extract was filtered in four batches and each was processed on a 1-L Ni Sepharose column that was scaled up 100-fold from the lab experiments. All column buffers contained 20 mM sodium phosphate, 0.5 M NaCl and 2 M urea and were at pH 8. Binding buffer contained 20 mM imidazole, wash buffer contained 50 mM imidazole and elution buffer contained 0.5 M imidazole. A 10-cm diameter column containing 1 L Ni-Sepharose 6 Fast Flow was equilibrated using 2 L of binding buffer at a volumetric flow rate of 200 mL/ min. The filtrate solution (~45 L) containing the fusion protein was then loaded into the column at the same flow rate. The column was washed with 8L binding buffer followed with a 4L wash buffer. Finally, the fusion protein was eluted with 8L elution buffer. When this step was completed, the column was rinsed and equilibrated with binding



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Page 26 of 32

buffer to prepare for the next batch. HPLC analysis of the column fractions from the four column batches showed a total recovery of 55.8 g intact heterodimeric fusion protein with peak area ratios of intact/truncated fusion protein ranging from 4.47 to 4.60 and peak area ratios of hetero/homodimers ranging from 2.86 to 3.25. Cyanogen bromide (CNBr) cleavage of the fusion protein The reaction was carried out using the fusion protein solution directly obtained from the Ni-purification column. The crude solution was transferred to a glass reactor and acidified to pH 1.5 with 1N HCl to afford a mixture that approximately contained 12 g crude protein per liter. CNBr (120 equivalent) was added relative to the amount of cleavable fusion protein as determined by HPLC. The resulting mixture was kept at 25 oC under nitrogen, stirred at 300 rpm, and periodically monitored by HPLC to maximize methionine cleavage while minimizing product degradation. It is extremely important to take appropriate precaution to avoid exposure to the toxic CNBr and the reaction byproduct MeSCN. For this reason, the reactor was fitted with a bubbling exhaust to a quench solution containing a 1:2 mixture of 1N NaOH and commercial grade bleach cooled with an external ice bath. Cleavage of fusion protein by CNBr was completed after 17 h. Removal of toxic MeSCN and excess CNBr was accomplished by distillation into the hypochlorite solution at 25 °C and confirmed by GC analysis. Following the distillation, the desired disulfide-bridged peptide was obtained in 50% yield along with undesired byproducts of homoserine side reaction.33 Chromatography of the CNBr cleavage reaction mixture: Isolation of disulfide-bridged peptide The CNBr reaction mixture was chromatographed on a Prochrom dynamic axial compression column (length 4.5 cm, internal diameter 4.5cm, Novasep) packed with Luna C18 10µm stationary phase and eluted with a gradient of 0.1 M ammonium acetate - acetonitrile mobile phase at 25°C and monitored by UV. Fractions containing the disulfide-bridged peptide were collected. Disulfide-bridged peptide


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containing fractions from several injections were combined. After lyophilization, disulfide-bridged peptide 1.8g was obtained corresponding to a chromatography yield of 80%. HPLC showed >99 AP. LC-MS and other physical data and comparison with authentic material prepared via solid phase peptide synthesis confirmed the identity of the disulfide-bridged peptide. ASSOCIATED CONTENT Supporting Information (SI) The supporting information is available free of charge on the ACS publication website at Cloning, expression, and production of individual chains, Fermentation and isolation of heterodimeric protein, NMR of disulfide-bridged peptide.

AUTHOR INFORMATION Corresponding author: *E-mail: [email protected] Notes The authors declare no competing financial interest.

REFERENCES 1. Bates, E., Bode, C., Costa, M., Gibson, C. M., Granger, C., Green, C., Grimes, K., Harrington, R., Huber, K., Kleiman, N. et al (2008) Intracoronary KAI-9803 as adjunct to primary percutaneous coronary



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intervention for acute ST-segment elevation myocardial infarction. Circulation 117, 886-896. 2. Li, M. T. (2006) Pharmaceutical formulation. U.S. Pat. Appl. 2006/0153867 A1. 3. Grimes, K., and Chen, L. E. (2009) Method of treating acute ST-elevation myocardial infarction with a delta PKC antagonist. U.S. Pat. Appl. 2009/0318351 A1. 4. Kent S. (2010) Editorial - Origin of the chemical ligation concept for the total synthesis of enzymes (proteins). Peptide Sci. 94 (4), iv-ix. 5. Marston, F. A. O. (1986) The purification of eukaryotic polypeptides synthesized in Escherichia coli. Biochem. J. 240, 1-12. 6. Moks, T., Abrahmsen. L., Osterolf, B., Josephson, S., Ostling, M., Enfors, S-O., Persson, I., Nilsson, B., and Uhlen, M. (1987) Large-scale affinity purification of human insulin-like growth factor from culture medium of Escherichia coli. Bio/Technology 5, 379-382. 7. Ford, C. F., Suominen, I., and Glatz, C. E. (1991) Fusion tails for the recovery and purification of recombinant proteins. Protein Exp. Purif. 2, 95-107. 8. LaVallie, E. R., and McCoy, J. M. (1995) Gene fusion expression systems in Escherichia coli. Cur. Opin. Biotech. 6, 501-506. 9. Kim, D-Y., Shin, N-K., Chang, S-G., and Shin, H-C. (1996) Production of recombinant human glucagon in Escherichia coli by a novel fusion protein approach. Biotechnology Techniques 10, 669-672. 10. Nilsson, J., Stahl, S., Lundeberg, J., Uhlen, M., and Nygren, P-A. (1997) Affinity fusion strategies for detection, purification, and immobilization of recombinant proteins. Protein Exp. Purif. 11, 1-8. 11. Hearn, M. T. W., and Acosta, D. (2001) Applications of novel affinity cassette methods: use of peptide


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fusion handles for the purification of recombinant proteins. J.Mol. Recog. 14, 323-369. 12. Wang, C., Castro, A. F., Wilkes, D. M., and Altenberg, G. A. (1999) Expression and purification of the first nucleotide-binding domain and linker region of human multidrug resistance gene product: comparison of fusions of glutathione S-transferase, thioredoxin, and maltose-binding protein. Biochem. J. 338, 77-81. 13. Kapust, R. B., and Waugh, D. S. (1999) Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused. Protein Sci. 8, 1668-1674. 14. Houchuli, E., Dobeli, H., and Schacher, A. (1987) New metal chelate adsorbent selective for proteins and peptides containing neighboring histidine residues. J Chromatog. 411, 177-184. 15. Houchuli, E., Bannwarth, W., Dobeli, H., Gentz, R., and Stuber, D. (1988) Genetic approach to facilitate purification of recombinant proteins with a novel metal chelate adsorbent. Bio/Technology 1321-1325. 16. Lingquist, C., Breitholtz, A., Brink-Nilsson, H., Moks, T., Uhlen, M., and Nilsson, B. (1989) Immobilization and affinity purification of recombinant proteins using histidine peptide fusions. Eur. J. Biochem. 186, 563-569. 17. Terpe K. (2003) Overview of tag protein fusions: from novel molecular and biochemical fundamentals to commercial systems. Appl. Microb. Biotech. 60, 523-533. 18. Maroux, S., Baratti, J., and Desnuelle, P. (1971) Purification and specificity of porcine enterokinase. J. Biol. Chem. 246, 5031-5039. 19. Light, A., and Janska, H. (1989) Enterokinase (enteropeptidase): comparative aspects. Trends in Biochem. Sci. 14, 110-112.



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20. Hosfield, T., and Lu, Q. (1999) Influence of the amino acid residue downstream of (Asp)4Lys on enterokinase cleavage of a fusion protein. Anal. Biochem. 269, 10-16. 21. Liu, S. W., and Franceshini, T. J. (2000) High expression Escherichia coli expression vector. US Pat Appl 2000/6068991A. 22. Smith, B. J. (2002) Chemical cleavage of proteins at methionyl-x peptide bonds. The protein protocols handbook, 2nd Edition (Walker, J. M., Ed.) pp 485-491, Humana Press, Totowa, NJ. 23. Landisch, M. R., and Kohlmann, K. L. (1992) Recombinant human insulin. Biotech. Progress 8, 469-478. 24. Sorensen, H. P., Sperling-Petersen, H. U., and Mortensen, K. K. (2003) A favorable solubility partner for expression of recombinant streptavidin. Protein Exp. Purif. 32, 252-259. 25. Fontana, A. (1972) Modification of tryptophan with BNPS-Skatole (2-(2-nitrophenylsulfenyl)-3-methyl-3-bromoindelenine. Meth. Enzym. 25, 419-423. 26. Zhang, Y. B., Howitt, J., McCorckle, S., Lawrence, P., Springer, K., and Freimuth, P. (2004) Protein aggregation during overexpression limited by peptide extensions with large net negative charge. Protein Exp. Purif. 36, 207-216. 27. O’Shea, E. K., and Limb, K. J. (1993) Peptide Velcro design of a heterodimeric coiled coil. Curr. Biol. 3, 658-667. 28. Chang, H-C., Bao, Z-Z., Yao, Y., Tse, A. G. D., Goyarts, E. C., Madsen, M., Kawasaki, E., Brauer, P. P., Sacchettini, J. C., Nathenson, S. G.et al. (1994) A general method for facilitating heterodimeric pairing between two proteins: Application to expression of α and β T-cell receptor extracellular segments. Proc. Nat. Acad. Sci. U.S.A 91, 11408-11412.


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29. Moll, J. R., Ruvinov, S. B., Pastan, I., and Vinson, C. (2001) Designed heterodimerizing leucine zippers with a range of pIs and stabilities up to 10-15 M. Protein Sci. 10, 649-655. 30. Mason, J. M., and Arndt, K. M. (2004) Coiled coil domains: Stability, specificity and biological implications. ChemBioChem 5, 170-176. 31. Mason, J. M., Schmitz, M. A., Muller, K. M., and Arndt, K. M. (2006) Semirational design of Jun-Fos coiled coils with increased affinity: Universal implications for leucine zipper prediction and design. Proc. Nat. Acad. Sci. U.S.A. 103, 8989-8994. 32. Olaofe, O. A., Burton, S. G., Cowan, D. A., and Harrison, S. T. L. (2010) Improving the production of a thermostable amidase through optimizing IPTG induction in a highly dense culture of recombinant Escherichia coli. Biochem. Eng. J. 52, 19-24. 33. Kaiser, R., and Metzka, L. (1999) Enhancement of Cyanogen Bromide Cleavage Yields for Methionyl-Serine and Methionyl-Threonine Peptide Bonds. Anal. Biochem. 266, 1–8.



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Biotechnology based process for production of a disulfide-bridged peptide Animesh Goswami, Steven L. Goldberg, Ronald L. Hanson, Robert M. Johnston, Olav K. Lyngberg, Yeung Chan, Ehrlic Lo, Steven H. Chan, Nuria de Mas, Antonio Ramirez, Richard Doyle, Wei Ding, Mian Gao, Stanley Krystek, Changhong Wan, Yeoun jin Kim, Deepa Calambur, Mark Witmer, James W. Bryson

Individual Chain Approach MCSFNSYELGSL

Fermentation

MCSFNSYELGSL

Expression

MCYGRKKRRQRRR

CSFNSYELGSL

Fusion protein Fermentation

MCYGRKKRRQRRR

PKC Cleavage

CYGRKKRRQRRR

AAMCYGRKKRRQRRR MCSFNSYELGSL

MCSFNSYELGSL Fermentation

Disulfide bond formation

AAMCYGRKKRRQRRR Fusion protein

Heterodimeric approach


Disulfide bond formation

TAT

Fusion protein

Expression

Expression in a single strain

Cleavage

AAMCYGRKKRRQRRR MCSFNSYELGSL Cleavage Disulfide-bridged fusion protein

CSFNSYELGSL CYGRKKRRQRRR Disulfide-bridged peptide

Biotechnology Based Process for Production of a ... - ACS Publications

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