Protocol for high-throughput cloning, expression, purification, and evaluation of bispecific antibodies

Li, Danqing; Partin, Alexander C.; Zhao, Liangjun; Chen, Irwin; Michaels, Mark L.; Wang, Zhong Lin; Garcés, Fernando; Gong, Danyang; Riley, Timothy P.

doi:10.1016/j.xpro.2022.101428

Cited by 2 publications

(2 citation statements)

References 15 publications

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Section: Discussionmentioning

confidence: 86%

“…This situation can be avoided by ordering codon optimised synthetic gene inserts without any internal Bsa I from suppliers such as TWIST or IDT. As we and others have found the Golden Gate method is particularly well suited to the direct cloning of synthetic gene fragments (22). For the system described in this work provided the synthetic genes are ordered with the above mentioned golden gate extensions (5’-AAAAAA GGTCTC AC ATG -target- TAATGA- CTCGA GAGACC AAAAAA-3’) the received insert(s) can then be used directly in a Golden Gate reaction with the vector of choice.…”

Section: Discussionmentioning

confidence: 95%

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A selection of Golden Gate vectors to simplify recombinant protein production inEscherichia coli

Fairhead,

Koekemoer,

Williams

et al. 2024

Preprint

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In this work a total of 22 Escherichia coli vectors have been created to allow the simple cloning (via Golden Gate) of the same DNA insert into multiple plasmids. This approach rapidly generates not only his, twin-strep and avi tagged constructs but also a variety of fusion proteins including the commonly used SUMO, MBP, GST and sfGFP. Each vector contains two BsaI sites allowing the Golden Gate cloning of inserts under the control of the powerful T7 promoter. This allow for construct expression in the standard protein production strain of E. coli BL21[DE3]. To further simplify use all vectors encode kanamycin resistance. We have evaluated the vectors using a test super folder GFP insert and found that using the Golden Gate process allows cloning efficiencies of greater than 90% to be routinely obtained. This allows the cloning and evaluation of many vector insert combinations to be achieved in a short time frame. The plasmid vector set described herein should prove useful to any investigator who has to routinely evaluate numerous protein expression constructs. These vectors are freely available on request or via Addgene.

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Section: Discussionmentioning

confidence: 86%

Section: Discussionmentioning

confidence: 95%