Design of peptide ligands for affinity purification of human growth hormone

Chandra, Divya; Timmick, Steven M.; Goodwine, Chaz; Vecchiarello, Nicholas; Shastry, Divya G.; Mullerpatan, Akshat; Trasatti, John P.; Cramer, Steven M.; Karande, Pankaj

doi:10.1002/jctb.6029

Cited by 8 publications

(6 citation statements)

References 42 publications

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“…The fluorescent dye here has an excitation wavelength of 495 nm and emission wavelength of 519 nm. The synthesis followed the conventional deprotection‐activation‐reaction‐capping scheme as mentioned elsewhere . Post synthesis, the amino acid protecting groups were removed and the peptides were cleaved from the solid support using a cocktail of solvents comprising trifluoroacetic acid, water, triisopropyl silane, and 2,2′‐(ethylenedioxy)diethanethiol.…”

Section: Methodsmentioning

confidence: 99%

Development of phage biopanning strategies to identify affinity peptide ligands for kappa light chain Fab fragments

Nascimento

Mullerpatan

Azevedo

et al. 2019

Biotechnology Progress

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In this work, two phage biopanning strategies were developed to identify affinity peptides for a single Fab and multiple kappa Fabs. For the biopanning rounds, protein L beads were employed to bind Fab targets in a fixed orientation, and NHS functionalized magnetic beads were used to facilitate evaluation of low pH elution conditions. The resulting peptide sequences were synthesized and the binding to different Fabs was evaluated using fluorescence polarization. The first biopanning approach yielded a peptide with similar affinities for two forms of the Fab (recombinantly expressed and post papain‐digestion) as well as the intact antibody. While moderate affinity was observed toward a murine variant of the Fab with the same complementarity determining regions (CDR) region but different framework, minimal binding occurred to a Fab with high sequence homology but containing different CDR loops. The second biopanning strategy yielded a peptide with affinity for all three kappa Fabs indicating that it may be a good lead for the development of more general affinity reagents for recombinant kappa Fabs. Finally, an affinity peptide column was developed, and its efficacy was demonstrated for Fab purification from a complex cell culture fluid mixture. The results presented in this article demonstrate that different peptide‐based phage biopanning strategies can be effectively employed to identify affinity peptide leads for specific Fab and more general kappa Fab purifications.

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Section: Methodsmentioning

confidence: 99%

Development of phage biopanning strategies to identify affinity peptide ligands for kappa light chain Fab fragments

Nascimento

Mullerpatan

Azevedo

et al. 2019

Biotechnology Progress

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“…The ACQUITY UPLC Protein BEH (Ethylene Bridged Hybrid) C 4 column was designed to separate large molecular weight proteins and has shown excellent performance in separating many challenging proteins, overcoming the shortcomings of 100% silica-based materials [27][28][29][30][31]. Among the glutenin fraction, HMW-GS are more hydrophilic and larger than LMW-GS.…”

Section: Optimization Of Rp-uplc Conditionsmentioning

confidence: 99%

A Rapid, Reliable RP-UPLC Method for Large-Scale Analysis of Wheat HMW-GS Alleles

et al. 2021

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High-molecular-weight glutenin subunits (HMW-GS) account for only 10% of total wheat storage proteins, but play an important role in the processing quality of wheat flour. Therefore, identifying HMW-GS alleles associated with good end-use quality provides important information for wheat breeders. To rapidly, accurately and reproducibly identify HMW-GS, we established an optimized reversed-phase ultra-performance liquid chromatography (RP-UPLC) method. Separation parameters were optimized using an ACQUITY UPLC Protein BEH C4 column and stepwise ACN gradient, and the separation patterns and retention times (RTs) of 22 subunits were comparatively analyzed in 16 standard wheat cultivars. All HMW-GS proteins were well separated within about 5.5 min, and all analyses were complete within 12 min. We distinguished the 16 subunits based on RT, although three subunits in 1Bx (1Bx7/1Bx7OE and 1Bx17) and three subunits in 1By (1By8*, 1By9 and 1By15) had overlapping RTs; these were differentiated by SDS-PAGE. To distinguish 1Bx7 and 1Bx7OE, which differ in protein abundance, RP-UPLC was combined with PCR analysis of DNA junction markers. The optimized method was successfully applied to determine HMW-GS alleles in a large collection of bread wheat germplasm (1787 lines). This protocol is an appropriate option for selecting lines harboring favorable HMW-GS alleles in wheat breeding.

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“…Affinity fusion-tag based purification of recombinant proteins has long been a simple and widely used technique to produce recombinant proteins at laboratory scale and for research use. 1 The use of generic affinity chromatography steps has tremendous potential to simplify protein production in a wide variety of settings from basic research to high-throughput structural biology. 2 However, owing to technical and economic reasons, such protein tagging technology has not yet been fully leveraged for proteins intended to be used as therapeutics.…”

Section: Introductionmentioning

confidence: 99%

Advanced purification platform using circularly permuted caspase‐2 for affinity fusion‐tag removal to produce native fibroblast growth factor 2

Lingg

Cserjan‐Puschmann

Fischer

et al. 2021

J of Chemical Tech & Biotech

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BACKGROUND: Recombinant proteins produced for use as biopharmaceuticals need to harbor their native N-terminus. A drawback in expression of recombinant proteins as fusion proteins with an affinity fusion-tag is that enzymatic or chemical processing is required to trim the artificial tag and release the true protein of interest. In many cases, however, this processing step generates an incorrect N-terminus.RESULTS: Human fibroblast growth factor 2 (FGF2) was expressed as a fusion protein in Escherichia coli fed-batch cultivations. The protein of interest (POI) carried an N-terminal affinity fusion-tag which enabled purification via affinity chromatography. After enzymatic removal of the affinity fusion-tag with a circularly permuted human caspase-2 (cpCasp2), the POI was further purified using subtractive affinity chromatography. Mass spectrometric analysis confirmed the authentic N-terminus of the POI. The generated POI was highly pure with 42 ppm host cell protein, 3.7 ∼g mL −1 dsDNA and ∼ 1000 EU mL −1 endotoxin. Only a small number of E. coli host cell proteins were co-purified with the POI. Because of the high specificity of the novel protease cpCasp2, no off-target cleavage could be observed. CONCLUSION: Our findings demonstrate that cpCasp2 can be used for the production of native proteins using a fusionprotein process. This represents a first case study at large laboratory scale for the production of an industrially relevant protein. This technology constitutes the basis of a highly scalable cpCasp2-based platform fusion protein process (CASPON technology) purification platform.

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Design of peptide ligands for affinity purification of human growth hormone

Cited by 8 publications

References 42 publications

Development of phage biopanning strategies to identify affinity peptide ligands for kappa light chain Fab fragments

Development of phage biopanning strategies to identify affinity peptide ligands for kappa light chain Fab fragments

A Rapid, Reliable RP-UPLC Method for Large-Scale Analysis of Wheat HMW-GS Alleles

Advanced purification platform using circularly permuted caspase‐2 for affinity fusion‐tag removal to produce native fibroblast growth factor 2

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