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Optimizing Recombinant Protein Production for Domain Antibodies: Proof-of-Concept

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Recent studies in traumatic brain injury (TBI) have found a temporal window where therapeutics on the nanometer scale can cross the blood-brain barrier and enter the parenchyma. Developing protein-based therapeutics is attractive for a number of reasons, yet, the production

Recent studies in traumatic brain injury (TBI) have found a temporal window where therapeutics on the nanometer scale can cross the blood-brain barrier and enter the parenchyma. Developing protein-based therapeutics is attractive for a number of reasons, yet, the production pipeline for high yield and consistent bioactive recombinant proteins remains a major obstacle. Previous studies for recombinant protein production has utilized gram-negative hosts such as Escherichia coli (E. coli) due to its well-established genetics and fast growth for recombinant protein production. However, using gram-negative hosts require lysis that calls for additional optimization and also introduces endotoxins and proteases that contribute to protein degradation. This project directly addressed this issue and evaluated the potential to use a gram-positive host such as Brevibacillus choshinensis (Brevi) which does not require lysis as the proteins are expressed directly into the supernatant. This host was utilized to produce variants of Stock 11 (S11) protein as a proof-of-concept towards this methodology. Variants of S11 were synthesized using different restriction enzymes which will alter the location of protein tags that may affect production or purification. Factors such as incubation time, incubation temperature, and media were optimized for each variant of S11 using a robust design of experiments. All variants of S11 were grown using optimized parameters prior to purification via affinity chromatography. Results showed the efficiency of using Brevi as a potential host for domain antibody production in the Stabenfeldt lab. Future aims will focus on troubleshooting the purification process to optimize the protein production pipeline.

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Date Created
2019-05

ASU Jazz combos

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Date Created
2018-10-11

ASU Jazz combos

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Created

Date Created
2018-10-10

ASU Jazz combos

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Date Created
2018-10-02

Witching Hours

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Witching Hours is the debut studio album of Chicago-born, Phoenix-residing trumpet player John Michael Sherman. It is a consummation of his work in the Arizona State University jazz studies program both as a performer and composer. Featured on the album

Witching Hours is the debut studio album of Chicago-born, Phoenix-residing trumpet player John Michael Sherman. It is a consummation of his work in the Arizona State University jazz studies program both as a performer and composer. Featured on the album are several other musicians who John Michael played alongside throughout his tenure at ASU, including Chaz Martineau on tenor saxophone, Evan Rees on piano, Reid Riddiough on guitar, Vince Thiefain on bass, Matt McClintock on drums, and Dan Meadows on baritone saxophone. The album features seven pieces, all original compositions or arrangements. The first track, "Workin' My Nerves", is a blues shuffle in the key of F. This is followed by "Scarborough Fair", an arrangement of the classic English folk tune in a rock style. The title track, "Witching Hours", is an cadaverous linear composition in 7/4 which is followed by "Goliath", a pseudo-tone poem about the biblical giant. "I Should Have Known" is a pensive ballad featuring an a capella intro and cadenza, followed by the most recent composition, a minor blues-esque piece entitled "Who Said That?" The final track, "Don't Change A Thing", is an upbeat samba which was written in John Michael's first year of college. These pieces demonstrate an understanding of the jazz tradition and exhibit influences from such musicians as Clifford Brown, Freddie Hubbard, Wayne Shorter, and Snarky Puppy. The album was recorded at Tempest Recording in Tempe and produced by Clarke Rigsby. Clarke is a veteran recording engineer and is the first choice of many of Phoenix's finest jazz musicians, including thesis director and head of the ASU jazz department Michael Kocour. The pieces were composed and recorded under the guidance of Mike Kocour and Jeff Libman. Witching Hours represents a culmination of John Michael's course in the Arizona State University jazz department and his endeavors as a trumpet player and composer.

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Date Created
2017-05