Quantifying vitamin K-dependent holoprotein compaction caused by differential γ-carboxylation using high-pressure size exclusion chromatography

Vanderslice, Nicholas C.; Messer, Amanda S.; Vadivel, Kanagasabai; Bajaj, S. Paul; Phillips, Martin; Fatemi, Mostafa; Xu, Weijie; Velander, William H.

doi:10.1016/j.ab.2015.03.019

Cited by 2 publications

(4 citation statements)

References 53 publications

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“…The Stokes radius of proteins is generally determined by analytical ultracentrifugation and is not available for most proteins. 16 …”

Section: Resultsmentioning

confidence: 99%

“…Why then are log M w versus elution volume plots so widely used? The Stokes radius of proteins is generally determined by analytical ultracentrifugation and is not available for most proteins …”

Section: Resultsmentioning

confidence: 99%

“…The Stokes radius of proteins is generally determined by analytical ultracentrifugation and is not available for most proteins. 16 Although the SEC behavior of asymmetric proteins is a scientifically valid concern, in reality it poses little problem with MASC. The amount of excluded protein in an SEC chromatogram of human serum (Figure 2) shows that <1% of the total protein in the serum will elute in the void volume with ASTP/analyte complexes.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

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Mobile Affinity Sorbent Chromatography

Kim

Regnier

2018

Anal. Chem.

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The objective in routine analyses is generally to determine a small number of analytes. With samples containing ~103 or more components there will be insufficient peak capacity to resolve analytes from nonanalytes. This issue was addressed herein through a new type of separation mechanism in which small groups of targeted analytes are bound with high affinity to a soluble analyte-sequestering transport phase (ASTP) composed of a ~25 nm Stokes radius hydrophilic polymer core (HPC). When introduced into a 30 nm pore diameter size-exclusion chromatography (SEC) column, ASTP/analyte complexes elute within minutes, together, unretained, and relatively pure in the first chromatographic peak. Nonanalytes, in contrast, enter pore matrices of the packing material, are retarded in elution velocity, and are eluted later, separated from analytes. Fabrication of ASTPs was achieved by covalently coupling an antibody or some other affinity selector to a high molecular weight HPC. Beyond sequestering analytes, the function of ASTPs is to act as a molecular weight shifting agent, conveying an effective molecular weight to analytes that is much larger than that of nonanalytes and causing them to elute in the SEC void volume. This mode of separation is referred to as mobile affinity sorbent chromatography (MASC). Subsequent to their purification, ASTP/analyte complexes were detected by fluorescence spectrometry.

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“…The Stokes radius of proteins is generally determined by analytical ultracentrifugation and is not available for most proteins. 16 …”

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: ■ Results and Discussionmentioning

confidence: 99%

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Mobile Affinity Sorbent Chromatography

Kim

Regnier

2018

Anal. Chem.

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“…The gamma-carboxylglutamic acid (Gla) content 5 27 28 of rFIX and other VKD coagulation proteins generate compaction in holoprotein structure needed for biological activity 29 . Thus, we used high pressure size exclusion chromatography (HPLC-SEC) to isolate functional rFIX from differentially carboxylated subpopulations that were nonfunctional.…”

Section: Resultsmentioning

confidence: 99%

Engineering protein processing of the mammary gland to produce abundant hemophilia B therapy in milk

Zhao

Ross

et al. 2015

Sci Rep

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Both the low animal cell density of bioreactors and their ability to post-translationally process recombinant factor IX (rFIX) limit hemophilia B therapy to <20% of the world’s population. We used transgenic pigs to make rFIX in milk at about 3,000-fold higher output than provided by industrial bioreactors. However, this resulted in incomplete γ-carboxylation and propeptide cleavage where both processes are transmembrane mediated. We then bioengineered the co-expression of truncated, soluble human furin (rFurin) with pro-rFIX at a favorable enzyme to substrate ratio. This resulted in the complete conversion of pro-rFIX to rFIX while yielding a normal lactation. Importantly, these high levels of propeptide processing by soluble rFurin did not preempt γ-carboxylation in the ER and therefore was compartmentalized to the Trans-Golgi Network (TGN) and also to milk. The Golgi specific engineering demonstrated here segues the ER targeted enhancement of γ-carboxylation needed to biomanufacture coagulation proteins like rFIX using transgenic livestock.

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Quantifying vitamin K-dependent holoprotein compaction caused by differential γ-carboxylation using high-pressure size exclusion chromatography

Cited by 2 publications

References 53 publications

Mobile Affinity Sorbent Chromatography

Mobile Affinity Sorbent Chromatography

Engineering protein processing of the mammary gland to produce abundant hemophilia B therapy in milk

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